Mez medical virology

Medical Virology
Mohamed Ezzat El Zowalaty, Ph.D.
Email:
elzow001@gmail.com
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Copyright© Dr. Mohamed El Zowalaty
By

Declaration and acknowledgment
I, Mohamed El Zowalaty, PhD would like to express special thankfulness to
the
• US CDC Public Health Image Library
• DHS/NIH/NIAID
• University of Minnesota
• All additional resources
for in education by providing images used in this presentation.
All images and photomicrographs used in this presentation were used for
teaching and educational purposes.
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Notice
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No parts of this copyrighted work
are allowed to be reproduced
without written permission from
author.
Any unauthorized use of this work
and its contents partly or wholly by
copying, printing, or distribution is
strictly prohibited and may be
unlawful.

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Lecture 1

General overview
Practical virology
 Laboratory Biosafety
 Virus diagnosis
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Learning Objectives
At the completion of the course, you will be able to:
 Identify the Biosafety Guidelines
 The safe use of Biological Safety Cabinets
 Describe the increased requirement of laboratory biosafety
 Compare and contrast laboratory safety procedures for BSL‐ 1 and BSL‐2
 Locate the safety devices available in the molecular Virology laboratory and use
properly
 Determine specimen types used for virus isolation, antigen detection and viral
molecular diagnostic tests
 Demonstrate cell culture techniques for maintenance and virus propagation
 Demonstrate virus propagation in hens’ eggs
 Demonstrate virus quantification methods
 Demonstrate molecular methods of PCR and real time PCR for the detection of
viral NAs
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• The information in this course is presented
with a combination of text and graphics.
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Laboratory methods in virology
 Imaging techniques
• Electron Microscopy
• Inverted microscopy
• Fluorescent microscopy
 Virus infectivity techniques
• Live virus isolation, propagation
• Virus quantification (Plaque assay and TCID50).
 Viral serology
• ELISA
• CFT
• VNT
 Molecular virology
• PCR, RT-PCR
• real time PCR, RT-qPCR
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Lab Diagnosis of Viral Infections
Virus culture and
isolation
-CPE
-Hemagglutination
-Plaque assay
-TCID50 assay
Detection of viral
antibody
-Hemagglutination
-inhibition test
-EIA/ELISA
Detection of viral
antigen
Immunofluorescence
-EIA/ELISA
-Western Blot
Immunoprecipitation
Detection of viral
genome
-(PCR)
-southern &
northern blot
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Safety Regulations and Good Microbiological Practice
• The use of good laboratory practice is an important factor in
safeguarding the health and safety of laboratory personnel.
• Viruses (like other micro-organisms) should be treated with
caution.
• A good aseptic technique is absolutely essential when dealing
with viruses. Not only does it safeguards you from infection
but prevents your cultures from becoming contaminated by
the surrounding bacteria
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International biohazard symbol and the
legend “biohazard” or “biohazardous waste”
BSL 1
BSL 2
BSL 3
BSL 2
Signage and Labels
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Biohazardous materials
• Biohazardous materials are infectious agents or other biological materials
that present a risk or potential risk to the health of humans, animals or
the environment.
• Biohazardous materials include:
• Microorganisms and viruses infectious to humans, animals or plants (e.g.
parasites, viruses, bacteria, fungi, prions, rickettsia) cultured human and
animal cells
• Certain types of recombinant DNA
• Biologically active agents that may cause disease in other living organisms
or cause significant impact to the environment or community. (i.e. toxins,
allergens, venoms)
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Hazard Communication Signs
• The Hazard Communication Sign is not intended to prohibit
access, but to communicate that biohazardous materials may
be present in the laboratory.
• Biohazard Labels
All equipment and materials used to manipulate, store, or
transport infectious material must be labeled with the universal
"Biohazard" symbol. The label must be orange or red-orange
with the biohazard symbol in a contrasting color.
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A Hazard is a
potential source of
harm or adverse
health effect on a
person
Risk is the likelihood (chance)
high or low that a person may
be harmed or suffers adverse
health effects if exposed to a
hazard.
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WHAT IS A RISK ASSESSMENT?
It is required that users of laboratories and those who control
workplaces to any extent must:
• identify the hazards in the workplaces under their control
• assess the risks presented by these hazards
A hazard is something with the potential to cause harm (for
example, chemical substances, machinery or methods of work),
while measuring the risk depends on:
• the likelihood of that harm occurring in the workplace
• the potential severity of that harm (the degree of injury or ill
health following an accident)
• the number of people who might be exposed to the hazard
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• BS officers must write down these workplace risks and what
to do about them.
• This is known as a risk assessment.
• Assessing risk means you must examine carefully what, in the
workplace (laboratory) , could cause harm to personnel using
the lab, other employees and other people, including
customers, visitors and members of the public. This allows
you to weigh up whether you have taken enough precautions
or whether you should do more to prevent harm.
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WHAT IS A RISK ASSESSMENT?

Risk Assessment
• A Risk Assessment is the best method for determining the proper
Biosafety Level and safety practices for your work.
• It should be based on the agent(s) and manipulations of the agent
that you will perform.
• Risk Groups have been assigned to biohazardous agents. They
indicate how dangerous is a particular bacteria, virus, or other
biohazard.
• First, determine the risk group of the agent(s) with which you are
working.
• Determine the recommended biosafety level by referring to one or
more of the updated biosafety resources.
• Determine if there are any manipulations that would indicate using
a higher level of containment such as: high titers/concentrations,
use of sharps, large volumes (>10 L) or creation of aerosols.
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Hierarchy of Control measures
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• Purpose: to reduce or eliminate exposure of individuals and
the environment to potentially hazardous biological agents.
• Achieved by implementing various degrees of
laboratory control and containment through:
• laboratory design
• access restrictions,
• personnel expertise and training,
• use of containment equipment,
• safe methods of managing
infectious materials in a laboratory
setting.
Background on biosafety
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Background on biosafety
• Pathogen Safety Data Sheets (PSDSs)
• https://my.absa.org/tiki-index.php?page=Riskgroups
• Pathogen Risk Group (RG) Classification
• Risk Group 1: Low individual and community risk
e.g Lactobacillus acidophilus, Saccharomyces cerevisiae, tobacco
mosaic virus
RG1 pathogens can be opportunistic and may pose a threat to
immunocompromised individuals.
• Risk Group 2: Moderate individual risk and low community risk
e.g Listeria monocytogenes, Campylobacter jejuni, Hepatitis A virus
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Biosafety (Cont’d)
• Risk Group 3: High individual risk and low community risk
e.g M. tuberculosis and High pathogenic avian influenza virus.
• Risk Group 4: High individual risk and high community risk
Ebola virus, Marburg virus, Herpes B virus
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Risk group
• In many countries, infectious agents are categorized in risk
groups based on their relative risk. Depending on the
country and/or organization, this classification system
might take the following factors into consideration:
• Pathogenicity of the organism
• Mode of transmission and host range
• Availability of effective preventive measures (e.g.,
vaccines)
• Availability of effective treatment (e.g., antibiotics)
• Other factor
• You may search the RG database at
https://my.absa.org/tikiindex.php?page=Riskgroups
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Risk groups
• It is important to understand that biological agents are classified in
a graded fashion such that the level of hazard associated with RG1
being the lowest and RG4 being the highest. EHS Biosafety follows
the NIH Guidelines categorization of Risk Groups as follows:
• RG1 – Are not associated with disease in healthy adult humans or
animals
• RG2 – Are associated with disease which is rarely serious and for
which preventative or therapeutics is often available
• RG3 – Are associated with serious or lethal human disease for
which preventative or therapeutics may be available
• RG4 – Are associated with lethal human disease for which
preventative or therapeutics are not readily available
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Biosafety and Biosafety Levels
• What is Biosafety?
Biosafety is the application of safety precautions that reduce a laboratorian’s risk of
exposure to a potentially infectious microbe and limit contamination of the work
environment and, ultimately, the community.
• What are Biosafety Levels (BSLs)?
There are four biosafety levels. Each level has specific controls for containment of
microbes and biological agents. The primary risks that determine levels of
containment are infectivity, severity of disease, transmissibility, and the nature of
the work conducted. Origin of the microbe, or the agent in question, and the route
of exposure are also important.
• Route of Exposure
• Route of exposure is the way a microbe gains access to a living organism. There are
four main routes of exposure
• Percutaneous, though broken or damaged skin
• Inhalation
• Mucous membranes of the eyes, nose, and mouth
• Ingestion
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Biosafety and Biosafety Levels
• The biosafety levels range
from BSL-1 to BSL-4. Each
biosafety level builds on
the controls of the level
before it. Every
microbiology laboratory,
regardless of biosafety
level, follows standard
microbiological practices.
The higher the containment level, the more stringent the safety measures.
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Biosafety Levels
• In contrast to Risk Groups, Biosafety Levels (BSL)
prescribe procedures and levels of containment for
the particular microorganism or material (including
Research Involving Recombinant or Synthetic Nucleic
Acid Molecules).
• Similar to Risk Groups, BSL are graded from 1 – 4.
• Detailed descriptions of containment practices and
biosafety levels can be found in the CDC-NIH
Guidelines Biosafety in Microbiological and
Biomedical Laboratories.
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Biosafety Levels
• A lab is designated as containment level 2 when its
features and practices allow the people working in it
to safely handle infectious agents that can be
transmitted by ingestion, inoculation and through
mucus membranes.
• In most cases, the agents handled in a containment
level 2 lab are not transmitted by the airborne route.
• The higher the containment level, the more stringent
the safety measures.
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Biosafety Levels
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• Each biosafety level has its own specific containment
controls that are required for the following:
• Laboratory practices
• Safety equipment
• Facility construction
Biosafety Levels
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BSL-1
If you work in a lab that is designated a BSL-1, the microbes there are not known to
consistently cause disease in healthy adults and present minimal potential hazard to
laboratorians and the environment. An example of a microbe that is typically worked
with at a BSL-1 is a nonpathogenic strain of E. coli.
Specific considerations for a BSL-1 laboratory include the following:
Laboratory practices
Standard microbiological practices are followed.
Work can be performed on an open lab bench or table.
Safety equipment
Personal protective equipment, (lab coats, gloves, eye protection) are worn as needed.
Facility construction
A sink must be available for hand washing.
The lab should have doors to separate the working space with the rest of the facility.
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BSL-2
BSL-2 builds upon BSL-1. If you work
in a lab that is designated a BSL-2, the
microbes there pose moderate
hazards to laboratorians and the
environment. The microbes are
typically indigenous and associated
with diseases of varying severity. An
example of a microbe that is typically
worked with at a BSL-2 laboratory
is Staphylococcus aureus
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BSL-2, continued
In addition to BSL-1 considerations, BSL-2 laboratories have the following containment
requirements:
Access to the laboratory is restricted when work is being conducted.
Safety equipment
Appropriate personal protective equipment (PPE) is worn, including lab coats and
gloves. Eye protection and face shields can also be worn, as needed.
All procedures that can cause infection from aerosols or splashes are performed
within a biological safety cabinet (BSC).
An autoclave or an alternative method of decontamination is available for proper
disposals.
The laboratory has self-closing doors.
A sink and eyewash are readily available.
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BSL-3
• BSL-3 builds upon the containment requirements of BSL-2.
• If you work in a lab that is designated BSL-3, the microbes there can be
either indigenous or exotic, and they can cause serious or potentially
lethal disease through respiratory transmission. Respiratory transmission
is the inhalation route of exposure.
• One example of a microbe that is typically worked with in a BSL-3
laboratory is Mycobacterium tuberculosis, the bacteria that causes
tuberculosis.
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BSL-3, continued
In addition to BSL-2 considerations, BSL-3 laboratories have the following containment
requirements:
Laboratorians are under medical surveillance and might receive immunizations for
microbes they work with.
Access to the laboratory is restricted and controlled at all times.
Safety equipment
Appropriate PPE must be worn, and respirators might be required.
All work with microbes must be performed within an appropriate BSC.
A hands-free sink and eyewash are available near the exit.
Exhaust air cannot be recirculated, and the laboratory must have sustained directional
airflow by drawing air into the laboratory from clean areas towards potentially
contaminated areas.
Entrance to the lab is through two sets of self-closing and locking doors
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• This photograph
suggests a BSL-3
laboratory. The
laboratorian is working
within a BSC and is
wearing a powered air
purifying respirator,
gloves, and a solid-
front gown
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BSL-4
• Builds upon the containment requirements of BSL-3 and is the
highest level of biological safety. There are a small number of
BSL-4 labs in the United States and around the world.
• The microbes in a BSL-4 lab are dangerous and exotic, posing
a high risk of aerosol-transmitted infections.
• Infections caused by these microbes are frequently fatal and
without treatment or vaccines. Two examples of microbes
worked with in a BSL-4 laboratory include Ebola and Marburg
viruses.
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BSL-4, Continued
In addition to BSL-3 considerations, BSL-4 laboratories
have the following containment requirements:
Change clothing before entering.
Shower upon exiting.
Decontaminate all materials before exiting.
Safety equipment
All work with the microbe must be performed within an
appropriate Class III BSC, or by wearing a full body, air-
supplied, positive pressure suit.
The laboratory is in a separate building or in an isolated
and restricted zone of the building.
The laboratory has dedicated supply and exhaust air, as
well as vacuum lines and decontamination systems.
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• NIH/CDC have defined 4 Biosafety Levels. Biosafety Levels (BSL)
are levels of containment (1 lowest, 4 highest containment.)
• BSL 1 - represents a basic level of containment that relies on
standard microbiological practices with no special primary or
secondary barriers recommended, other than a sink for hand
washing.
• Examples of organisms in BSL-1 include non-pathogenic
laboratory strains of Escherichia coli, Serratia marcescens,
Staphylococcus epidermidis.
• BSL-1 agents includes recombinant DNA activities using such
non-pathogenic organisms as hosts for the expression of genes
incorporated into bacterial plasmids or low risk viral vectors
such as baculovirus or adeno associated virus.
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Containment
• “Containment“: safe methods for managing infectious
materials in the laboratory environment where they are being
handled or maintained.
• The purpose of containment is to reduce or eliminate
exposure of laboratory personnel and the outside
environment to potentially hazardous agents.
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Containment Laboratories
• Microorganisms and viruses are divided into 4 Biosafety
Levels (BSL) by the US CDC.
• Generally the containment level and risk group are the same.
That is, an RG2 pathogen would be handled in CL2, but not
always – there are some exceptions.
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Elements of containment
laboratory practice and technique
safety equipment
facility design.
The risk assessment of the work to be done with
a specific agent will determine the appropriate
combination of these elements.
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BSL-1 laboratory
Figure 1. A typical BSL-1 laboratory.
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BSL-1 laboratory
• BSL 2 - is suitable for work involving agents of moderate
potential hazard to personnel and the environment.
• Many BSL-2 pathogens are opportunistic, meaning they
don’t ordinarily cause disease in healthy human adults, but
may cause disease in children and immunocompromised
adults.
• Examples of organisms handled in BSL-2 include
Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus
cereus, Klebsiella pneumonia, Proteus vulgaris,
Streptococcus pyogenes, and Salmonella typhimurium.
• Many viruses fall in BSL 2 category such as Low Pathogenic
avian influenza strains, Rotavirus, and measles
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BSL-2 laboratory
Figure 2. A typical Biosafety Level 2 laboratory. Procedures likely to generate
aerosols are performed within a biological safety cabinet.
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BSL-3 laboratory
• BSL 3 - is suitable for work involving indigenous or exotic agents which
may cause serious or potentially lethal disease as a result of exposure by
inhalation.
The laboratory is separated from general traffic flow and accessed through an
anteroom (double door entry or
basic laboratory – Biosafety Level 2) or an airlock.
An autoclave is available within the facility for decontamination of wastes
prior to disposal. A sink with hands-free operation is available.
Inward directional airflow is established and all work with infectious materials
is conducted within a biological safety cabinet.
Examples of BSL-3 agents include Mycobacterium tuberculosis and Bacillus
anthracis, High pathogenic avian Influenza virus H5N1 , MERS-CoV.
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BSL-3 laboratory
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BSL-4 laboratory
• BSL 4 - required for work with dangerous and
exotic agents that pose a high risk of aerosol-
transmitted laboratory infections and life-
threatening disease.
• Examples of BSL-4 agents include Ebola virus,
Marburg virus, and Lassa fever virus.
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BSL-4 laboratory
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View into the decontamination
chamber of a BSL4 laboratory Highly
restricted area
access door to
BSL4
laboratory
A fully suited
researcher in a BSL4
laboratory
Negative pressure room for suiting up before
entering the BSL4 laboratory
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RISK
GROUP
BIOSAFET
Y LEVEL
LABORATORY
TYPE
LABORATORY
PRACTICES
SAFETY
EQUIPMENT
1 Basic- Basic teaching, GMT No11e; openbench
Biosafety research work
Level 1
2 Basic - Primary health GMT plus protective Open bench plus.
BSC
Biosafety services; diagnostic clothing, biohazard for potential aerosols
Level 2 services' research sign
3 Containment - Special diagnostic As Level 2 plus BSC and/or other
Biosafety services, research special clothing, primary devices for all
Level 3, controlled access, activities
directional airflow
4 Maximum
containment
Dangerous
pathogen units
As Level 3 plus
airlock entry, shower
Class Ill BSC, or
positive pressure suits
Biosafety exit, special waste in conjunction with
Level 4 disposal Class II BSCs, double
ended autoclave
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Biological safety cabinet
• The biological safety cabinet, or BSC, is a type of
primary containment equipment designed to protect
you from exposure to infectious material or toxins, to
prevent loss of containment and to protect your
specimens from contamination.
• Other types of primary containment devices include
isolators, centrifuges with sealable cups, process
equipment, fermenters, microisolator cages, ventilated
cage racks and sealed biological waste containers. Use
each type of primary containment devices for its
intended use only – each has its own limitations.
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Fume hoods, BSCs, and Laminar flow devices
• Chemical fume hoods are familiar to most lab workers.
• Although BSCs are often called “hoods” and some
classes of BSCs function similarly to chemical fume
hoods, don’t confuse the two. Chemical fume hoods
provide protection from toxic chemicals, but no
protection from infectious material or toxins.
• BSCs are sometimes confused with laminar flow
devices or “clean benches” that are designed to protect
samples from airborne contamination. They direct air
towards the operator, so you should never use one for
handling infectious materials.
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Fume Hoods vs Biosafety
Cabinets
Fume Hoods Biosafety Cabinets
•No filtration of air
•Exhausts chemical fumes
outside the laboratory
•Suitable for chemicals and non-
sterile work
• Never used for infectious
agents
•HEPA filtration of air intake and
exhaust
•Recirculates filtered air in to
laboratory
•ensure sterility
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Biosafety Practices, not allowed
• HEPA filters are fragile, and
can lose their efficiency if
they are exposed to:
• Solvent vapours
• Heat (from open flame or
other heat source)
• Intense vibrations (from
moving the BSC)
• No storage is allowed inside BSC
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maintain the air curtain
Clean complete work BSC
One at a
time
UV before
and after use
BSC Practices, recommended
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BSCs provide:
• Effective primary containment for work with infectious material or
toxins whose primary route of infection is inhalation.
• Personnel and environmental protection when working with high
concentrations (e.g., pure cultures) or large volumes of infectious
material or toxins.
• A certified BSC is to be used for procedures that:
• May produce infectious aerosols or aerosolized toxins, when
aerosol generation cannot be contained through other methods
(for example, opening tubes, using an inoculating loop, pipetting,
centrifugation, mixing and homogenizing, needles and syringes,
pouring infectious material);
• Involve high concentrations or large volumes of infectious material
or toxins.
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Selection of a Safety Cabinet through Risk Assessment
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Types of Biological safety cabinets
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The Class II Biological Safety Cabinet (BSC)
Offers 4 types of protection
1) Personal
2) Product
3) Cross contamination
4) Environmental
64
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Basic Anatomy of a BSC
Exhaust Hepa Filter
Aperture
Downflow Hepa Filter
Downflow Fan
Exhaust Fan
70%
30%
65
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Airflow – Balance
Ambient air
Clean air
Potentially hazardous air
Exhaust air
Downflow AirInflow air
66
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Airflow Balance – The Importance
INFLOW
Too High!
DOWNFLOW
Too High!
67
BALANCED
Inflow and Downflow
#1 #3 #2
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External Airflow Effects Your BSC
Thermo Scientific Herasafe KS biological safety cabinet
Place BSC away from foot traffic
Place BSC away from AC units
Move slowly and gently in and out the work area
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Using Class II, Type A2 BSC
• Principle
• Start clean
• Stay clean
• Minimize airflow disturbance
• Minimize border crossings
• Organize movement within BSC
work area
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10
Using Your Class II, Type A2 BSC
• Start by Preparing
Turn it on when you are getting ready to work in the
cabinet.
Don your appropriate PPE; lab coats, gloves and so on.
Wipe down the BSC work surface.
Place a plastic backed absorbent towel on the work
surface to reduce spatter and facilitate clean-up in the
event of a spill.
Gather your supplies, wipe them down and load them
into the cabinet.
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• Organize the work area from clean to dirty as shown.
Clean Dirty
(for right handed user)
71
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Clean Dirty
(for right handed user)
72
• When finished
Safely dispose of waste
Wipe down BSC work area
Close the window
After 5 minutes, turn off the BSC
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Features to Help Protect You and Your Samples
• Smart Flow
• Designed to ensure a safe working
environment, even in between
certifications
• Independent control of inflow/exhaust
and downflow
• No need for manual airflow control
of a damper
• Exhaust motor automatically compensates
in response to HEPA loading
• Downflow motor compensates in
response to exhaust motor
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Comparison of Biosafety Cabinet Characteristics
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Comparison of Biosafety Cabinet Characteristics
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• In high containment zones, a certified BSC or other
appropriate primary containment device is to be
used for:
• All activities involving open vessels of infectious
materials and toxins.
• BSCs are not required in containment areas where
the room itself serves as the primary containment
(i.e. animal cubicles), or when working with large
sized-animals, as personnel would be wearing the
appropriate personal protective equipment (PPE).
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Keep It In
Check
The certification procedure should include:
Halogen leak test to insure the positive pressure
air flow plenums are gas tight
Measurement of air inflow velocity
Measurement of the airflow within the cabinet to
assure it is uniform and unidirectional
A leak test of the HEPA filter to verify proper
installation and that it is leak-free
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• When maintained and used properly, a BSC is an effective
primary containment barrier.
• It is not a substitute for good laboratory practice and proper
technique.
• A BSC is only effective if it is maintained and used properly.
• A BSC will not contain aerosols if it is used incorrectly, or if the
worker uses poor technique.
• BSCs should not be used as a chemical fume hood.
• BSCs do not protect the user from toxic fumes.
– However, there are different types of BSCs and you can use
a Class II Type B2 (hard ducted) with small amounts of
volatile toxic chemicals or radionuclides.
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Biosafety guidelines
• Give strict attention to all instructions and if not clear, check
with your instructor or the technical staff before undertaking
any experiment.
• Each individual embarking on these, or any other, activities is
responsible for his or her own safety and also for the safety of
others affected by their work
• Refer to BIOM 324 Medical Virology Laboratory manual for
detailed description of biosafety guidelines.
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Standard Microbiological Practices
• Standard microbiological practices are those
practices that are common to all laboratories. These
practices may include
• Not eating, drinking, or applying cosmetics in the lab
• Washing hands after working with infectious
materials and before leaving the lab
• Routinely decontaminating work surfaces
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Personal Protective Equipment (PPE)
• Equipment worn to
minimize exposure to
a variety of hazards.
Examples of PPE
include laboratory
coats, gowns, gloves,
eye protection, face
shields, shoe covers,
and respirators.
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BSL‐1 Personal Protection
• Wear safety goggles or safety glasses
when handling liquid cultures, when performing procedures
that may create a splash hazard, or when spread plating.
• Wear closed-toe shoes that cover the top of the foot.
• Wear gloves when the student’s hands have fresh
cuts or abrasions, when staining microbes, and when handling
hazardous chemicals.
• Gloves are not required for standard laboratory procedures
(BSL-1) if proper hand hygiene is performed.
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BSL‐1 Physical Space Requirements
Personal Protection
• Goggles/safety glasses, closed-toe shoes, gloves are all required for BSL-2 work.
• Recommended BSL-1/ Required BSL-2: Wear laboratory coats.
Require all laboratory space to include:
• Nonporous floor, bench tops, chairs/stools
• Sink for hand washing
• Eyewash station
• Lockable door to the room
• Follow proper pest control practices
Recommended/Required BSL-2:
• Personal belongings kept separate from the work area
Recommended/Required BSL-2:
• Use a working and validated autoclave
• Required BSL-2: Biohazard Signage
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Standard Lab Practices
Standard:
• NO eating/eating/applying cosmetics
• Closed toe shoes, long pants
• Disinfection of work area before and after working in lab
• Labels on ALL containers
• NO mouth pipetting – do not to generate aerosols
• NO electronic devices will be removed from lab.
• Spill cleanup – inform lab instructor, follow lab standard
• BSL-1Recommended/BSL-2 required:
• Note-taking separate from workspace
• Electronic notebooks.
• Writing implements stay in the lab.
• Recommended BSL-1/BSL-2 required:
• Microincinerators or disposable loops
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One of documented Real-life events
• Biosafety level 2 (BSL‐2) work, students should
be competent performing BSL‐1 activities.”
• Notes from the field: Salmonella typhimurium
infections associated with a community
college microbiology laboratory —Maine, USA
(2013)
Morbidity and Mortality Weekly Report (MMWR) November 1, 2013 / 62(43);863‐863
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One of documented Real-life events
• •Either non‐pathogenic or attenuated bacterial
strains should be used when possible, especially in
teaching laboratories. This practice will help reduce
the risk of students and/or their family members
becoming ill.
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Investigation of incident
• In interviews, ill persons answered questions about different exposures in
the week before becoming ill.
• Eighteen (86%) of 21 ill persons interviewed reported being enrolled in
either a human biology course or microbiology course.
• Fifteen (83%) of these 18 ill persons were students, and three (22%) were
employees.
• Many ill persons reported several behaviors while they were working in
the laboratory that would increase the risk of acquiring a Salmonella
infection, including not wearing gloves or lab coats, lack of handwashing,
and using the same writing utensils and notebooks outside of the
laboratory. Additionally, many ill persons did not recall receiving
laboratory safety training.
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Today’s lab Assignment
Your turn: Exercise 1
Select the biosafety level described by the conditions of the following example. Levels
are listed below. A microbiology graduate student is working on a project under the
following conditions: Work is conducted on a standard laboratory table or bench. A
nonpathogenic laboratory strain of E. coli is being used. Minimal PPE, such as a lab
coat, gloves, and eye protection might be worn but are not necessary.
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Exercise 2
Select the biosafety level shown in the photo below.
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Exercise 3
Do you believe the biosafety level at which you are operating
should be adjusted?
A. No, I believe my initial choice was accurate.
B. Yes, my understanding has changed.
C. I am uncertain now.
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Exercise 4
Two people shouldn’t work in a BSC at the same time. Can you think of
some reasons why not? (Select all that apply.)
A. The increased arm movement of two people working in a BSC at once may
cause disruptions across the fragile air curtain.
B. The 'break-point', where the half the air splits and goes to the front and
back of the BSC, is pushed farther back when there are two people.
C. Excess materials inside the cabinet increases the risk of contamination of
samples.
D. Increased risk of contamination results from an increase in the amount of
movement of material and equipment entering and exiting as well as within
the cabinet.
E. It will lead to more rapid failure of the HEPA filter.
F. Two people working at once create a crowded work area.
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Exercise 5
Which of the following can be used when working with biohazardous
material?
A. Fume hood
B. Biological Safety Cabinet
C. Laminar flow hood
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Lecture 2
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Laboratory Diagnosis of Viral
Infections
Virus culture and
isolation
-CPE
-Hemagglutination
-Plaque assay
-TCID50 assay
Detection of viral
antibody
-Hemagglutination
-inhibition test
-EIA/ELISA
Detection of viral
antigen
Immunofluorescence
-EIA/ELISA
-Western Blot
Immunoprecipitation
Detection of viral
genome
-(PCR, RT-PCR,
qPCR)
-southern &
northern blot
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Virus isolation
and
propagation

Study of Viruses
• The study of viruses is known as virology.
• Viruses can be studied using two experimental approaches.
• The first approach is through isolation and cultivation, and the
second approach is through detection, identification and
diagnosis.
• For isolation and cultivation, animals, plants, chicken embryo,
and tissue culture are used.
• For detection, identification and diagnosis, there are several
methods. These methods include tissue culture methods,
physical methods, serological methods, immunological
methods, and molecular biology methods.
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Virus culture
• Viruses are very small, non-cellular strict intracellular agents that
must infect host cells to propagate and replicate.
• Outside host cell: inert particles
• Inside host cell: live virion (parasite)
• Virus multiply only in living cells .
• Outside its host cell a virus is an inert particle
• Why virus is considered as inert biochemical complex?
because:
1. It does not replicate outside living cell.
2. Viruses do not respire nor move, nor grow but do reproduction.
3. For a virus to multiply it must infect a permissive host cell.
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Virus Isolation and Cultivation
• Animals and Chick Embryo:
Laboratory animals and chick embryo were the first methods
that were used to cultivate virus.
• The method of virus culture in animals and CE is rarely used as
it is not convenient. However, when preparing for bulk virus,
(e.g. antigen or vaccine production) the usage of chick embryo
is useful. However most laboratories use nowadays cell
culture methods for virus propagation.
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Virus Isolation and Cultivation
• Inoculation of laboratory animals is used when some viruses
can only be isolated using this method.
• Normally, mice and monkeys are the laboratory animals that
are used.
• Mice are the most widely employed animals in virology. The
different routes of inoculation in mice are intracerebral,
subcutaneous, intraperitoneal or intranasal.
• Signs of disease or death in animals are observed after
inoculation. By testing for neutralization of their pathogenicity
for animals by standard sera, viruses can be identified.
• LD50 is determined for viruses using this method.
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Primary Isolation of viruses
• Generally three methods are employed for the virus
cultivation:
1. Inoculation of virus into animals, e.g. Poliovirus culture in
monkeys.
2. Inoculation of virus into embryonated hens’ eggs, e.g.
Influenza virus culture
3. Propagation of viruses in cell cultures, e.g. Influenza virus
culture in MDCK cells
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Primary Isolation of viruses
• Animals, plants and epithelial cells of membranes within
embryonated eggs have been used extensively to culture
viruses.
• Cells must therefore be available either within living host
systems, in vivo, or as primary or immortalized cells cultured
in the laboratory, in vitro for culture of viruses.
• Because of differences in cellular tropism between viruses, a
single cell type for virus isolation for diagnostic purposes is
not available
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Introduction to virus isolation
• Investigation of a new virus will start with attempts to its
isolation and culture is a permissive cell.
• Cell culture remains integral with virology, as viruses are
obligate intracellular parasites that require replication within
a living cell to produce copies of themselves (i.e., to form
progeny virions).
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Introduction to virus isolation
• Both animal and plant cells are propagated in cell cultures.
• The only other practical alternatives to cell culture are to
propagate the viruses in susceptible animal or plant hosts.
• E.g. Propagation of Poliovirus in monkeys.
• Propagation of Tobacco Mosaic Virus in plants. (Q)
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In ovo virus culture
• Embryonated chicken eggs constitutes a transitional state
between in vivo and in vitro virus production.
• Influenza virus was one of the first viruses to be produced
using this technique.
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Fertile eggs are incubated at 37oC in presence of 60% humidity in incubator and
candled daily. At the age of 9-11 day-old, ECEs are used for in ovo virus culture by
allantoic route.

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CK Embryo stages
Courtesy MEZ_U of Minnesota_2009

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Routes of virus inoculation

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Egg Candling

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Egg Candling

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Egg Candling

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In ovo virus inoculation

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Allantoic fluid Harvest

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“…., virus interested scientists will not fully comprehend
viruses unless have viruses “isolated” in hand ,….”
Mohamed El Zowalaty, Emory University, USA

Lecture 3
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Virus isolation and Propagation
Cell culture

Cell culture
History
• Tissue culture was devised at the beginning of the
twentieth century [Harrison, 1907; Carrel, 1912].
• Reprogramming of adult cells to become
pluripotent stem (iPS) cells (Yu et al. 2007)
• Induction of iPS cells by reprogramming with
valproic acid (Huangfu et al. 2008)
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Tissue Culture Applications
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Advantages of Tissue culture
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Limitations
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Tissue/cell culture
• Cell culture is still the most common method for the propagation of viruses.
• Types of Tissue culture
a. Organ culture
b. Primary explant culture
c. Cell culture
• Cell culture is defined as the removal of cells from their host tissue (an
animal, insect, or plant) and their subsequent growth in a favorable artificial
environment ( in vitro).
• Cells may be removed from the host tissue directly and disaggregated by
enzymatic or mechanical means before cultivation, or they may be derived
from a previously established cell line or cell strain that has already been
established
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• Primary culture
• It refers to the stage of the culture after the cells are isolated
from the tissue and proliferated under the appropriate
conditions until they occupy all of the available substrate (i.e.,
reach confluence). At this stage, the cells have to be
subcultured (i.e., passaged) by transferring them to a new
vessel with fresh growth medium to provide more room for
continued growth.
• Subculturing, also referred to as passaging, is the removal of
the medium and transfer of cells from a previous culture into
fresh growth medium, a procedure that enables the further
propagation of the cell line
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• Cell line
• After the first subculture, the primary culture becomes known
as a cell line or subclone. Cell lines derived from primary
cultures have a limited life span (i.e., they are finite), and as
they are passaged, cells with the highest growth capacity
predominate, resulting in a degree of genotypic and
phenotypic uniformity in the population
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Developing a cell line
Live tissue ( e.g. CK femurs)
cells to be cultured
Culture vessel with
appropriate growth media
Cell adhere to vessel and
grow to form a monolayer
Lift cells into solution
with enzyme
Seed cells into new
culture vessels
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• Finite vs continuous cell line
• Normal cells usually divide only a limited number of times
before losing their ability to proliferate, which is a genetically
determined event known as senescence; these cell lines are
known as finite.
• However, some cell lines become immortal through a process
called transformation, which can occur spontaneously or can
be chemically or virally induced. When a finite cell line
undergoes transformation and acquires the ability to divide
indefinitely, it becomes a continuous cell line (immortalized).
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Fundamentals of cell culture
Cell culture conditions
Culture conditions vary widely for each cell type, but the artificial
environment in which the cells are cultured invariably consists of
a suitable vessel containing the following:
• Substrate or medium that supplies the essential nutrients
(amino acids, carbohydrates, vitamins, minerals)
• Growth factors hormones
• Gases (O2, CO2)
• Regulated physico-chemical environment (pH, osmotic
pressure, temperature)
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• Monolayer culture signifies that the cells are grown attached
to the substrate.
• Anchorage dependence means that attachment to (and
usually some degree of spreading onto) the substrate is a
prerequisite for cell proliferation.
• Monolayer culture is the mode of culture common to most
normal cells, with the exception of hematopoietic cells.
• Suspension cultures are derived from cells that can survive
and proliferate without attachment (anchorage independent);
this ability is restricted to hematopoietic cells and
transformed cell.
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• Types of cell culture systems
• There are two basic systems for growing cells in culture, as
monolayers on an artificial substrate (i.e., adherent culture)
or free-floating in the culture medium (suspension culture)
• The majority of the cells derived from vertebrates, with the
exception of hematopoietic cell lines and a few others, are
anchorage-dependent and have to be cultured while attached
to a solid or semi-solid substrate (adherent or monolayer
culture) on a suitable substrate that is specifically treated to
allow cell adhesion and spreading (i.e., tissue-culture
treated).
• However, many cell lines can also be adapted for suspension
culture and can be grown floating in the culture medium
(suspension culture). Similarly, most of the commercially
available insect cell lines grow well in monolayer or
suspension culture
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• A monolayer culture is an anchorage-
dependent culture. It grows attached to the
surface of a flask.
• A suspension culture is an anchorage-
independent culture. Suspension cultures
consist of rounded cells floating in medium
or lightly adhered to the flask. Some
suspension cultures form floating aggregates.
• It is possible to have a third, mixed culture
which contains both flat, adherent cells and
floating, rounded cells in the medium.
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Cell culture

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Adherent Cell Culture Suspension Cell Culture
Appropriate for most cell types, including
primary cultures
Appropriate for cells adapted to suspension
culture and a few other cell lines that are
nonadhesive (e.g., hematopoietic)
Requires periodic passaging, but allows easy
visual inspection under inverted microscope
Easier to passage, but requires daily cell
counts and viability determination to follow
growth patterns; culture can be diluted to
stimulate growth
Cells are dissociated enzymatically (e.g.,
TrypLE™ Express, trypsin) or mechanically
Does not require enzymatic or mechanical
dissociation
Growth is limited by surface area, which may
limit product yields
Growth is limited by concentration of cells in
the medium, which allows easy scale-up
Requires tissue-culture treated vessel Can be maintained in culture vessels that are
not tissue-culture treated, but requires
agitation (i.e., shaking or stirring) for adequate
gas exchange
Used for cytology, harvesting products
continuously, and many research
applications
Used for bulk protein production, batch
harvesting, and many research applications
Characteristics of adherent and suspension cell cultures

• Most mammalian cells in culture can be divided in to
three basic categories based on their shape and
appearance (i.e., morphology) and certain cells display
morphological characteristics specific to their specialized
role in host.
• Fibroblastic (or fibroblast-like) cells are bipolar or
multipolar, have elongated shapes, and grow attached to
a substrate. e.g. BHK-21 (ATCC® CCL-10™)
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Morphology of mammalian cells in culture

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Epithelial-like cells are polygonal in shape with more regular
dimensions, and grow attached to a substrate in discrete
patches e.g. HeLa, MDCK, and Vero cells (ATCC® CCL-81™)

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Lymphoblast-like cells are spherical in shape and usually
grown in suspension without attaching to a surface e.g. Daudi
cells (ATCC® Number: CCL-213™), THP-1 (ATCC® TIB-202™),
and Sf9 cells (ATCC® Number: CRL-1711™)
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• Successful cell culture depends heavily on keeping the cells free
from contamination by microorganisms such as bacterial, fungi,
and viruses.
• Non sterile supplies, media, and reagents, airborne particles
laden with microorganisms, unclean incubators, and dirty work
surfaces are all sources of biological contamination.
• Aseptic technique, designed to provide a barrier between the
microorganisms in the environment and the sterile cell culture,
depends upon a set of procedures to reduce the probability of
contamination from these sources.
• The elements of aseptic technique are a sterile work area, good
personal hygiene, sterile reagents and media, and sterile
handling.
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Contamination of cell cultures is easily the most common
problem encountered in cell culture laboratories, sometimes
with very serious consequences.
Cell culture contaminants can be divided into two main
categories:
• Chemical contaminants such as impurities in media, sera,
and water, endotoxins, plasticizers, and detergents.
• Biological (viable) contaminants such as bacteria, molds,
yeasts, viruses, mycoplasma, as well as cross
contamination by other cell lines.

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Cell culture environment:
• Cell culture environment affects cell growth and cell culture
conditions vary for each cell type.
• One of the major advantages of cell culture is the ability to
manipulate the physico- chemical (i.e., temperature, pH,
osmotic pressure, O2 and CO2 tension) and the physiological
environment (i.e., hormone and nutrient concentrations) in
which the cells propagate. With the exception of temperature,
the culture environment is controlled by the growth media.

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Culture media
Animal cell culture media is a complex mixture and must provide organic and
inorganic nutrients at the right levels for specific cell type. There are three basic
classes of media which differ in their requirement for supplementation with
serum.
Serum is vitally important as a source of growth and adhesion factors, hormones,
lipids and minerals for the culture of cells in basal media. In addition, serum also
regulates cell membrane permeability and serves as a carrier for lipids, enzymes,
micronutrients, and trace elements into the cell.
However, using serum in media has a number of disadvantages including
• high cost, problems with standardization,
• specificity, variability, and unwanted effects such as stimulation or inhibition
of growth and/or cellular function on certain cell cultures.
• If the serum is not obtained from reputable source, contamination can also
pose a serious threat to successful cell culture experiments

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Basal (traditional) media, majority of cells will grow in basal
media once serum is added, and contain amino acids, vitamins,
inorganic salts, and carbon source, but these media must be
supplemented with serum to be a complete medium.
Reduced serum media, contain reduced amount of serum.
Serum free media, different from basal media and is designed
for cell growth and avoid the disadvantages of serum in media,
Advantages of SFM;
• Ability to make the medium selective for specific cell types by
choosing the appropriate combination of growth factors
• Precise evaluation of cellular functions
• Better control over physiological response

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pH level:
Most normal mammalian cell lines grow well at pH 7.4, and
there is very little variability among different cell strains.
However, some transformed cell lines have been shown to
grow better at slightly more acidic environments (pH 7.0 – 7.4),
and some normal fibroblast cell lines prefer slightly more basic
environments (pH 7.4 – 7.7).
Insect cell lines such as Sf9 and Sf21 grow optimally at pH 6.2.

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CO2 level
Usually, 4 – 10% CO2 is common for most cell culture experiments.
The growth medium controls the pH of the culture and buffers the
cells in culture against changes in the pH. Usually, this buffering is
achieved by including an organic (e.g., HEPES) or CO2-bicarbonate
based buffer. Because the pH of the medium is dependent on the
delicate balance of dissolved carbon dioxide (CO2) and
bicarbonate (HCO3–), changes in the atmospheric CO2 can alter
the pH of the medium.

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Optimal temperature for cell culture largely depends on the
body temperature of the host from which the cells were isolated.
Most human and mammalian cell lines are maintained at 36°C
to 37°C for optimal growth.
Insect cells are cultured at 27°C for optimal growth; they grow
more slowly at lower temperatures and at temperatures
between 27°C and 30°C.
Avian cell lines require 38.5°C for maximum growth

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Morphology check of mammalian cell
cultures
• Cellular morphology refers to the optical observation of a
magnified cell culture.
• This can be the simplest and most direct method used to
identify the state of cells.
• Obtaining morphology information from comparative
observations both at high and low culture densities
depends on knowledge of several factors.
• Morphology can vary between cell lines depending on the
health of the cells and, in some cases, the differentiation
state

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• Morphology can change with plating density as well as
with different media and sera combinations. Cell
morphology is best monitored through frequent, brief
observations.
• In general, if a culture has an unusual appearance, there is
likely a problem. It is recommended that researchers be
alert during periodic morphology checks and maintain cell
morphology images for comparisons.

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• It is important to become very familiar with the shapes,
structures and overall appearance of healthy mammalian
cells in culture.
• Being familiar with uninfected cells lays the basis for all
future observations of virus infected cells.
• Cell monolayers, one cell thick are cultured from a small
number of cells which are initially seeded onto a tissue
culture non porous solid surface; these cells adhere to the
plastic surface and begin to divide.
• Division continues until the progeny cells meet another
cell on the surface. At this stage cell division stops a
phenomenon known as contact inhibition. Eventually a
continuous (confluent) cell monolayer is produced

• Morphology is an indication of the health or identity of a
culture.
• Cell culture morphology can change in response to slight
variations in culture conditions.
• Cell density, medium, serum, pH, % CO2, incubator humidity
and the substrate of the vessels are a few of the factors that
can cause differences in morphology both among cultures and
among different areas of the same culture
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Cell cultures observation under the
inverted microscope
– The appearance of a cell monolayer
– The shape of individual cells
– The differences between the cells
– The presence of sub-cellular structures
– The cell growth on the plastic surface
– The color of the medium
– The clarity/cloudiness of the medium
– Confluence of cell monolayer
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Subculturing adherent cells
Prior to cell culture practice, it has to be remembered that
• cell culture practices are performed within the cell culture
laboratory which is specifically designed laboratory with
specific equipment,
• general safety laboratory precautions are to be strictly,
• Cell culture supplies should be available and sterile.
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Cell culture Passage

• Successful cell culture depends mainly on proper practices
and follow of aseptic procedures including maintaining the
following
– Sterile work area.
– Good personal hygiene Wash your hands before and after
working with cell cultures.
– Use of sterile reagents and media.
– Sterile handling.
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Cell culture hood layout

• In order to passage specific cell after acquiring the cell,
adherent cells, unlike suspension cells, must be removed from
the plastic surface before cell counting and cell passage.
• Such adherent cells are detached using an enzyme, such as
trypsin, and the process in sometimes referred to as
trypsinization.
• One flask of confluent cells can therefore be passaged into
multiple flasks containing fresh medium. Cell passage,
therefore, expands the overall number of cells and produces
more confluent cell monolayers.
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• Subculturing suspension cells is somewhat less complicated
than passaging adherent cells.
• Because the cells are already suspended in growth medium,
there is no need to treat them enzymatically to detach them
from the surface of the culture vessel, and the whole process
is faster and less traumatic for the cells
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Lecture 4
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Cell counting using a
haemocytometer
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Objective
• Cell counts are necessary in order to establish or monitor
growth rates as well as to set up new cultures with known cell
numbers.
• Hemocytometers are commonly used to estimate cell number
and determine cell viability.
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Hemocytometer
• A hemocytometer is a fairly thick glass slide with two counting
chambers, one on each side. Each counting chamber has a
mirrored surface with a 3 × 3 mm grid of 9 counting squares.
(as shown in figure 6).
• The chambers have raised sides that will hold a coverslip
exactly 0.1 mm above the chamber floor. Each of the 9
counting squares holds a volume of 0.0001 ml.
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Counting chamber: This one is called the Neubauer
improved.
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Hemocytometer
• The platform is engraved with lines longitudinal and
latitudinal (graticule, a network of fine lines in the focal plan
of the eyepiece of an optical instrument) which intersect to
make squares of known area.
• Two graticules are present on either side of central groove in
the platform
• The frame of the counting chamber consists of 9 large squares
each with a 1 mm2 area. As shown in the figure each graticule
covers an area of 9 mm2 and is divided into 9 squares 1 mm by
1 mm.
• Each corner square (4 in total) is divided into 16 smaller
squares 0.25 mm by 0.25 mm. These are the ones used to
count cells (WBCs). As shown in figure, the central square is
split in 25 squares.
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• Each one of the 25 central squares
is subdivided in 16 small squares..
These are used to count smaller
objects.
• As the area of a corner square
(1mm2) and the distance from the
coverslip to the platform (0.1 mm
or 0.2 mm) is known, the volume
of cell suspension above the
corner square can be calculated.
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Grid layout of the Neubauer Improved
hemocytometer.
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A haemocytometer counting chamber (grids)
2 grids/hemocytometer
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Haemocytometer arrangement and dimensions
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Example of 0.1 mm hemocytometer
• When we put the sample under the coverslip, the cell
suspension reaches a height of 0.1 mm. Taking these data into
account, and considering one of the large squares, the volume
will be:
0.1 mm (depth) x 1 mm (square length) x 1 mm (square width)
= 0.1 mm3
or 0.01 cm x 0.1 cm x 0.1 cm = 10-4 cm3
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Appearance of the haemocytometer grid visualised under
the microscope.
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Estimating cell density
To calculate number of cells, cells are counted to ensure
accuracy according to system shown in next figure.
Correcting for dilution: If the sample was diluted before
counting, then this must be taking into consideration as well. We
assume that the sample was diluted 1:10. The final result is
therefore n cells x 10 = N cells in 1 ml.
Averaging: If one did not count all of the cells in a large square
(1mm x 1mm) then it is necessary to average the results first
before proceeding.
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Counting cells in a hemacytometer to ensure of cells accuracy and consistency. Count
the cells within the large square and those crossing the edge on two out of the four
sides. (x; excluded cells, ü; counted cells, blue; dead cells; plain, live cells).
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Do not count cells on the top and right lines.
Here it’s necessary to count the in the big square because
there are too few cells in individual small squares.
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Calculation
• Count 4 corner squares and calculate the average.
• Each large square of the hemocytometer, with cover slip in
place, represents a total volume of 0.1 mm3 (1.0 mm X 1.0
mm X 0.1 mm) or 10-4 cm3.
• Since 1 mm3 is equivalent to approximately 1 ml, the total
number of cells per ml will be determined using the following
calculations:
• Average viable cell count per square = Total number of viable
cells in 4 squares / 4.
• Dilution Factor = Total Volume (Volume of sample + Volume
of diluting liquid) / Volume of sample.
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Lecture 5
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Observation of
Cytopathic effects of viruses
What does the virus do to the host cell?

Cytopathic effects
• Virus replication in animals, ECEs or cell culture leads to
several changes and consequences that include
1. Embryo death
2. Animal death
3. Cell death (cytocidal infection)
Cell death might or might not involve cell lysis (cytolysis)
depending on virus.
4. Cytopathology: cytopathic effects due to viral infections can
be sued to measure biological activity of many viruses.
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Definitions
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Cells that support viral replication are called permissive.
Infections of permissive cells are usually productive because
infectious progeny virus is produced. Most productive infections
are called cytocidal (cytolytic) because they kill the host cell.
Infections of nonpermissive cells yield no infectious progeny
virus and are called abortive.
When the complete repertoire of virus genes necessary for virus
replication is not transcribed and translated into functional
products the infection is referred to as restrictive.
In persistent and in some transforming infections, viral nucleic
acid may remain in specific host cells indefinitely; progeny virus
may or may not be produced.

Virus-host cell interactions
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Virus-host cell interactions may produce either
• Cytocidal (cytolytic) infections, in which production of new
infectious virus kills the cell;
• persistent infections, in which the virus or its genome
resides in some or all of the cells without killing most of
them;
• transformation, in which the virus does not kill the cell,
but produces genetic, biochemical, physiologic, and
morphologic changes that may result in the acquisition of
malignant properties.
• The type of virus infection and the virus-induced effects on
cells are dependent on the virus, the cell type and species,
and often the physiologic state of the cell.

Damage to the Host Cell and Persistent
Infections
• Cytopathic effects- virus-induced damage to
the cell that alters its microscopic appearance
• Inclusion bodies- compacted masses of viruses
or damaged cell organelles
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Morphologic and Structural Effects
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• Infection of permissive cells with virus leads to productive
infection and often results in cell death (cytocidal, cytolytic
infection). The first effects of the replication of cytocidal
viruses to be described were the morphologic changes
known as cytopathic effects.
• Cultured cells that are infected by most viruses undergo
morphologic changes, which can be observed easily in
unfixed, unstained cells by a light microscope. Some viruses
cause characteristic cytopathic effects; thus, observation of
the cytopathic effect is an important tool for virologists
concerned with isolating and identifying viruses from infected
animals or humans

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Development and progression of viral cytopathology. Human embryo skin muscle
cells were infected with human cytomegalovirus and stained at selected times to
demonstrate (A) uninfected cells, (B) late virus cytopathic effects (nuclear
inclusions, cell enlargement), (C) cell degeneration, and (D) a focus of infected cells
in a cell monolayer (i.e., a plaque).

Cytopathic effects
• Many types of cytopathic effects occur. Often the first sign of viral
infections is rounding of the cells.
• In some diseased tissues, intracellular structures called inclusion
bodies appear in the nucleus and/or cytoplasm of infected cells.
• Inclusion bodies were first identified by light microscopy in smears
and stained sections of infected tissues. Their composition can often
be clarified by electron microscopy. In an adenovirus infection, for
example, crystalline arrays of adenovirus capsids accumulate in the
nucleus to form an inclusion body.
• Inclusions may alternatively be host cell structures altered by the
virus. For example, in reovirus-infected cells, virions associate with
the microtubules, giving rise to a crescent-shaped perinuclear
inclusion.
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• Important for the diagnosis of viral infections
• Some viral infections maintain a carrier relationship
– The cell harbors the virus and is not immediately lysed
– Persistent infections- from a few weeks to the remainder
of the host’s life
• Some viruses remain in a chronic latent state,
periodically becoming activated
• Some viruses enter their host cell and permanently
alter its genetic material, leading to cancer
– Oncogenic viruses
– Their effect is called transformation
– Oncoviruses- mammalian viruses capable of initiating
tumors
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• Infection of cells by other viruses causes specific alterations in
the cytoskeleton of cells. For example, extensive changes in
cellular intermediate filaments in relation to formation of viral
inclusions may be observed after cytomegalovirus infection
• A particularly striking cytopathic effect of some viral infections
is the formation of syncytia, or polykaryocytes, which are
large cytoplasmic masses that contain many nuclei (poly,
many; karyon, nucleus) and are usually produced by fusion of
infected cells
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Low-power view of continuous line of Hep-2 cells infected with RSV
Syncytia
CPE

Morphological Changes
Inclusion bodies –
microscopically these are
visible sites of viral assembly
or cellular damage. They are
often used as a diagnostic
tool. Examples include:
 Virions in the nucleus
(Adenovirus)
 Virions in the
cytoplasm
(Rhabdovirus- Negri
bodies of rabies virus)
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Figure 6.16
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Formation of multinucleated cells. The figure represents the cytopathology of measles virus-induced syncytia.

Syncytia formation
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Figure 6.22
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Morphological changes
Viral protein associated with host microtubules (Reovirus)
Factories of viral replication in the cytoplasm (Poxvirus)
Clumps of ribosomes in capsids (Arenavirus)
Clumps of chromatin (herpesviruses)
Morphological alterations
Nuclear pyknosis (shrinking) (Picornaviruses)
Proliferation of membranes (Picornaviruses)
Proliferation of the nuclear membrane (Alphaviruses)
Formation of vacuoles in the cytoplasm (Papovaviruses)
Apoptosis (will discuss this more later)
Formation of syncytia (Paramyxoviruses and Coronaviruses) which
are giant, multinucleated cells formed by the fusion of plasma
membranes
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 Margination and breakage of chromosomes
(Herpesviruses)
 Rounding up and detachment of tissue culture cells
– due to apoptosis (Herpes and Rhabdoviruses)
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Cytopathic Effect
• Some viruses kill the cells in
which they replicate, and
infected cells may
eventually detach from the
cell culture plate.
• As more cells are infected,
the changes become visible
and are called cytopathic
effects.
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Rounding of infected cells, fusion with adjacent cells to form a syncytia and
ultimate lysis, as compared with non-infected cells. A: Non-infected cells; B: Cells
with cytopathic effects.

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Transmission of H1N1 influenza from infants to mother ferrets causes
upper and lower respiratory tract infection with significant pathology.
Paquette SG, Banner D, Huang SSH, Almansa R, Leon A, et al. (2015) Influenza Transmission in the Mother-Infant Dyad Leads to Severe Disease, Mammary Gland
Infection, and Pathogenesis by Regulating Host Responses. PLoS Pathog 11(10): e1005173. doi:10.1371/journal.ppat.1005173
http://journals.plos.org/plospathogens/article?id=info:doi/10.1371/journal.ppat.1005173
Green arrows denote dense
cell accumulation; black
arrows denote diffuse
immune cell infiltration.
Black arrows not included on
Day 7 adult tissue due to
widespread infiltration.

Fig 4. 2009 H1N1 transmission from mothers to infants
results in severe lower respiratory tract pathology.
Paquette SG, Banner D, Huang SSH, Almansa R, Leon A, et al. (2015) Influenza Transmission in the Mother-Infant Dyad Leads to Severe Disease,
Mammary Gland Infection, and Pathogenesis by Regulating Host Responses. PLoS Pathog 11(10): e1005173. doi:10.1371/journal.ppat.1005173
http://journals.plos.org/plospathogens/article?id=info:doi/10.1371/journal.ppat.1005173
Harvested lungs from control infants
and infants of inoculated nursing-
mothers were processed for
histopathological assessment.
Tissue morphology was assessed by
hematoxylin & eosin staining. Data
was collected from three
independent litter
inoculations/infections (3
inoculated/infected mothers, 16
infants, and 3 mock
inoculated/infected mothers) and
results are a representative of the
inoculations/infections. pmi = Post-
Mother-Inoculation
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Examples of Cytopathic Effects of Viral Infection
• Nuclear shrinking (pyknosis)
• Proliferation of nuclear
membrane
• Vacuoles in cytoplasm
• Syncytia (cell fusion)
• Margination and breaking
of chromosomes
• Rounding up and
detachment of cultured
cells
• Inclusion bodies
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Cytopathic Effect
Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells.
(Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)
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Cytopathic Effect
Syncytium formation in cell
culture caused by RSV (top), and
measles virus (bottom).
(courtesy of Linda Stannard, University of Cape
Town, S.A.)
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Haemadsorption
Syncytial formation caused by mumps virus and haemadsorption of
erythrocytes onto the surface of the cell sheet.
(courtesy of Linda Stannard, University of Cape Town, S.A.)
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 CPE is very rarely caused by a harmful protein with
no other purpose in the infective process.
 CPE is usually a secondary result of changes in the
host metabolism caused by viral replication.
 Viruses may halt or alter host cell DNA synthesis,
transcription, and/or protein synthesis
(translation)
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• Effects on Cell Physiology
• Research into the pathogenesis of virus infections suggests a
close correlation between cellular physiologic responses and
the replication of some viruses.
• Other virus-associated alterations in cell physiology are
related to insertion of viral proteins or other changes in the
cell membrane.
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Alteration of cytoskeleton organization by virus infection. Normal cells have
networks of microtubules, and intermediate filaments throughout the cytoplasm.
Infection with reovirus causes a perinuclear aggregation of microtubules, and infection
with cytomegalovirus causes a modification of intermediate filaments proteins,
including their relocation into the nuclear and cytoplasmic inclusion bodies.

Effects on Cell Biochemistry
• Genotoxic Effects
• Chromosome damage may be caused directly by the virus particle or
indirectly by events occurring during synthesis of new viral
macromolecules (RNA, DNA, protein). The chromosome damage (Fig. 44-
5) may or may not be faithfully repaired, and in either case, it may or may
not be compatible with survival of the infected cell. When the cell
survives, the virus genome may persist within the cell, possibly leading to
continued instability of cellular genomic material or to altered expression
of cellular genes (e.g., cellular oncogenes). Virus-induced genomic
instability appears to be associated with accumulation of mutations and
related to the process of cell immortalization and oncogenic
transformation
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Viruses that Infect Bacteria
• Bacteriophage
• Most contain dsDNA
• Often make the bacteria they infect more
pathogenic for humans
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T-even Phages
• Icosahedral capsid head containing DNA
• Central tube surrounded by a sheath
• Collar
• Base plate
• Tail pins
• Fibers
• Similar stages as animal viruses
– Adsorb to host bacteria
– The nucleic acid penetrates the host after being injected through a rigid
tube inserted through the bacterial membrane and wall
– Entry of the nucleic acid causes the cessation of host cell DNA replication
and protein synthesis
– The host cell machinery is then used for viral replication and synthesis of
viral proteins
– As the host cell produces new parts, they spontaneously assemble
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Figure 6.18
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Figure 6.19
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Lysogeny: The Silent Virus Infection
• Temperate phages- special DNA phages that undergo
adsorption and penetration but are not replicated or released
immediately
• Instead the viral DNA enters an inactive prophage stage
• Lysogeny: the cell’s progeny will also have the temperate
phage DNA
• Lysogenic conversion: when a bacterium acquires a new trait
from its temperate phage
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A few definitions…
Viral pathogenesis:
= process by which a virus causes disease
Virulence:
= capacity of a virus to cause disease
Viral disease:
= sum of the effects of
(1) the virus replication and direct damage to
cells
(cytopathogenesis)
plus (2) of the immune response on the host
(immunopathogenesis)
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Why study viral pathogenesis?
• The study of viral pathogenesis is intellectually engaging and fun
• Acquire knowledge on the molecular mechanisms by which
viruses cause disease
• to treat and prevent viral disease
– AIDS
– Rabies
– Hepatitis
– Influenza, etc…
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Avian influenza H5N1 isolate.
Image courtesy@ Dr. Mohamed Ezzat El Zowalaty 2009

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Lecture 6
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Cytopathic effects
• Virus replication in animals, ECEs or cell culture leads to
several changes and consequences that include
1. Embryo death
2. Animal death
3. Cell death (cytocidal infection)
Cell death might or might not involve cell lysis (cytolysis)
depending on virus.
4. Cytopathology: cytopathic effects due to viral infections can
be sued to measure biological activity of many viruses.
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Identification of Virus Isolates
• A more definitive viral diagnosis is carried out by further testing of the
viral isolate
• This can be achieved by performing:
– Infectivity assays
• A viral neutralization assay
– The application of immunoassay techniques such as:
• IF staining of infected cells
• ELISA
• Western blotting
– Molecular techniques
• nucleic acid
The application of these techniques are particularly useful for
detecting specific viral replication in cultures in the absence of a CPE
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Identification of Virus Isolates
• Not all viruses will produce a CPE and some viruses are slow
to replicate in cell culture.
• Immunoassay techniques can also allow early detection of
viral replication prior to the formation of a CPE and allow
more rapid viral diagnosis
• The availability of specific and sensitive monoclonal
antibodies directed against viral antigen has greatly enhanced
the use of these techniques in viral diagnosis
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Quantification of Viruses
• Involves counting the number of viruses in a specific volume
to determine the virus concentration
• Many approaches are available to determine the
concentration of viruses in a given tissue
– Infectivity assays,
– Molecular assays
– Direct counting of virus particles using electron microscopy
• These enable the virologist to calculate the number of
infectious viral particles per unit volume
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• It is utilized in many procedures including:
– Research
– Diagnosis of early viral infection
– Monitor a patient's response to anti-virus therapy
– Production situations where the quantity of virus at
various steps is an important variable
• For example, the production of viral vaccines
• recombinant proteins using viral vectors
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• The most commonly used methods to quantify viruses can be
subdivided into three broader categories:
– Techniques measuring viral infectivity
• TCID50
• Viral plaque assay
– Those that examine viral nucleic acid and protein
• qPCR (real time or quantitative PCR)
• Western blotting
– Immunoassyas
• ELISA
– Those that rely on direct counting of physical viral particles
• Viral flow cytometry
• Transmission electron microscopy
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TClD50 (Tissue Culture Infectious Dose)
• This is a quantal assay which determines the dilution of virus
required to infect or cause CPE in 50% of inoculated cell
cultures
• The assay can be carried out in culture tubes or 96-well
microtiter plates
• Different viral dilutions are prepared and inoculated on cell
culture
• The virus replicates and the progeny virus that is released into
the supernatant fluid is free to infect any other cell
• The cytopathological damage is allowed to develop usually
over a period of days (depending on the given virus and the
cells)
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Quantification of CPE
• Tissue Culture Infective Dose 50 (TCID50): a
measure of virulence of virus
• Why Quantify?
– Virulence
– Immunity
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Typically, the 50% infectious dose (TCID50 or tissue culture 50% infectious dose) is
calculated using a mathematical analysis of the data. Popular methods include that of
Reed and Munch, or that of Spearman and Karber.

Infectivity assays
• Quantal assays detect the presence of infectious virus by use
of an “all or none” approach or evidence for the presence or
absence of infection and not the amount of infection. Does a
tissue culture monolayer show CPE? Is an egg infected? Has
an animal died?
• Focal assays rely on the detection and counting of foci of
infection, e.g. a focus of CPE (plaque) or a focus of
inflammatory response (pock) which allows for the
quantitative determination of the number of infectious units
as opposed to the qualitative approach of the quantal assay.
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ID50
• Serial dilution of virus are inoculated into cell cultures,
embryonated chicken eggs, or suitable host plants or animals,
and the virus titre taken as the reciprocal of the dilution at
which 50% of the recipients are affected. These assays are
appropriately named as 50% tissue culture infectious doses
(TCID50) in which the titer reciprocal of the virus dilution
which kills 50% of the culture inoculated; and 50% lethal dose
assay (LD50) where the titre is reciprocal of the virus dilution
which affects 50% of the inoculated eggs.
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• What is TCID50 ?
• time consuming,
laborious
• inexpensive and
gives more
accurate results
than plaque
assay
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Determine the virus infectivity titre using the Reed and Muench (1938) method
for the determination of the 50% Tissue culture infectious dose (TCID50):
Two formulas are used:
i. Proportionate Distance =
(% CPE at dilution above 50%) – (50%)
(% CPE at dilution above 50%) – (% CPE at dilution below 50%)
ii. - Log = dilution above 50% CPE ratio (i.e. 10-3 would be -3)
iii. ((PD)+(-log(dilution interval))
iv. TCID50 = 10(ii + iii)
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TCID50 interpretation
• TCID50 is the tissue culture infectious dose which will infect
50% if the cell monolayers challenged with the defined
inoculum.
• If the titer is "103 TCID50/0.1 ml, MK, 2 days," it means that
when a 0.1 ml inoculum of a 1:1,000 dilution of the virus is
added to each of four tubes containing monkey kidney (MK)
cells, two tubes are expected to become infected.
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TCID50 Procedure
• Count wells exhibiting CPE
• Ideally you would know all the dilution
factors to get infection rates of zero to 100
percent
0
100
Decreasing Dilution
CPE
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Calculation of TCID50
• In any biological quantification, the most desirable
endpoint is one representing a situation in which half of
the inoculated animals or cells show the reaction (death
in the case of animals and CPE case of cells) and the
other half do not.
• Reed-Muench Method of computing a 50% endpoint of a
virus titration
• Calculates the proportionate distance between dilutions
which infect above and below 50% of the wells
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TCID50
10-4
1 2 3 4 5 6
10-1 10-5
10-6
10-7
Control
B
C
D
A
10-2
10-3
Dilution Infected %
Infected
10-1 3/3 100
10-2 3/3 100
10-3 3/3 100
10-4 2/3 66
10-5 1/3 33
10-6 0/3 0
10-7 0/3 0
PD = (66 – 50)
(66 – 33)
PD = 0.48
-Log dilution above 50% = 4 (from 10-4)
4(-Log)+ 0.48(PD)= 4.48
TCID50 = 104.48/0.1ml infection dose
TCID50 = 105.48/ml viral titer
CPE
No CPE
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Log of
virus
dilution
Infected
test units
Non infected
test units
Cumulative
infected (A)
Cumulativenon-
infected
(B)
Ratio of
A/(A+B)
%
Infected
-5 4 0 4 0 8/8 100
-6 3 1 4 1 4/5 80
-7 1 3 1 4 1/5 20
-8 0 4 0 8 0/8 0
Example
Using the following sample data calculate the infectivity titre of
the virus.
Infected samples using the TCID50 would be represented by those showing CPE,
quadruplicate wells were inoculated with each dilution and those showing CPE were
recorded as a fraction of that total.
The dilution that would correspond to the 50% endpoint (using the above table) lies
somewhere between the 10-6 (80% infected) and the 10-7 (20% infected) virus dilutions.
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