Download Lab 22 DNA-based Screening for Smallpox as a Bio

Lab 22 DNA-based Screening for
Smallpox as a Bio-defense Strategy
This information is from the CDC website
Smallpox Fact Sheet
The Disease
Smallpox is a serious, contagious, and
sometimes fatal infectious disease. There is
no specific treatment for smallpox disease,
and the only prevention is vaccination. The
name smallpox is derived from the Latin word
for “spotted” and refers to the raised bumps that appear on the face and body of an
infected person. See image
There are two clinical forms of smallpox. Variola major is the severe and most common
form of smallpox, with a more extensive rash and higher fever. There are four types of
variola major smallpox: ordinary (the most frequent type, accounting for 90% or more of
cases); modified (mild and occurring in previously vaccinated persons); flat; and
hemorrhagic (both rare and very severe). Historically, variola major has an overall
fatality rate of about 30%; however, flat and hemorrhagic smallpox usually are fatal.
Variola minor is a less common presentation of smallpox, and a much less severe
disease, with death rates historically of 1% or less.
Smallpox outbreaks have occurred from time to time for thousands of years, but the
disease is now eradicated after a successful worldwide vaccination program. The last
case of smallpox in the United States was in 1949. The last naturally occurring case in
the world was in Somalia in 1977. After the disease was eliminated from the world,
routine vaccination against smallpox among the general public was stopped because it
was no longer necessary for prevention.
Where Smallpox Comes From
Smallpox is caused by the variola virus that emerged in human populations thousands
of years ago. Except for laboratory stockpiles, the variola virus has been eliminated.
However, in the aftermath of the events of September and October, 2001, there is
heightened concern that the variola virus might be used as an agent of bioterrorism. For
this reason, the U.S. government is taking precautions for dealing with a smallpox
outbreak.
Transmission
Generally, direct and fairly prolonged face-to-face contact is required to spread
smallpox from one person to another. Smallpox also can be spread through direct
contact with infected bodily fluids or contaminated objects such as bedding or clothing.
Rarely, smallpox has been spread by virus carried in the air in enclosed settings such
as buildings, buses, and trains. Humans are the only natural hosts of variola. Smallpox
is not known to be transmitted by insects or animals.
A person with smallpox is sometimes contagious with onset of fever (prodrome phase),
but the person becomes most contagious with the onset of rash. At this stage the
infected person is usually very sick and not able to move around in the community. The
infected person is contagious until the last smallpox scab falls off.
You may want to explore What We Learn About Smallpox from Movies - Fact or Fiction
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CDC Fact Sheet on Smallpox
Smallpox Disease
Incubation Period
(Duration: 7 to 17 days)
Not contagious
Initial Symptoms
(Prodrome)
(Duration: 2 to 4 days)
Sometimes
contagious*
Early Rash
(Duration: about 4 days)
Most contagious
Rash distribution:
Exposure to the virus is followed by an incubation period during which people
do not have any symptoms and may feel fine. This incubation period averages
about 12 to 14 days but can range from 7 to 17 days. During this time, people
are not contagious.
The first symptoms of smallpox include fever, malaise, head and body aches,
and sometimes vomiting. The fever is usually high, in the range of 101 to 104
degrees Fahrenheit. At this time, people are usually too sick to carry on their
normal activities. This is called the prodrome phase and may last for 2 to 4 days.
A rash emerges first as small red spots on the tongue and in the mouth.
These spots develop into sores that break open and spread large amounts of the
virus into the mouth and throat. At this time, the person becomes most
contagious.
Around the time the sores in the mouth break down, a rash appears on the skin,
starting on the face and spreading to the arms and legs and then to the hands
and feet. Usually the rash spreads to all parts of the body within 24 hours. As the
rash appears, the fever usually falls and the person may start to feel better.
By the third day of the rash, the rash becomes raised bumps.
By the fourth day, the bumps fill with a thick, opaque fluid and often have a
depression in the center that looks like a bellybutton. (This is a major
distinguishing characteristic of smallpox.)
View enlarged
image.
Pustular Rash
(Duration: about 5 days)
Contagious
Pustules and
Scabs (Duration:
about 5 days)
Contagious
Resolving Scabs
(Duration: about 6 days)
Contagious
Scabs resolved
Not contagious
Fever often will rise again at this time and remain high until scabs form over the
bumps.
The bumps become pustules—sharply raised, usually round and firm to the
touch as if there’s a small round object under the skin. People often say the
bumps feel like BB pellets embedded in the skin.
The pustules begin to form a crust and then scab.
By the end of the second week after the rash appears, most of the sores have
scabbed over.
The scabs begin to fall off, leaving marks on the skin that eventually become
pitted scars. Most scabs will have fallen off three weeks after the rash appears.
The person is contagious to others until all of the scabs have fallen off.
Scabs have fallen off. Person is no longer contagious.
* Smallpox may be contagious during the prodromal phase, but is most infectious during
the first 7 to 10 days following rash onset
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Case Study
World Health Organization (WHO) initiated a worldwide vaccination campaign to
eradicate smallpox. This program resulted in the steady decline of the virus with the last
naturally occurring case in Somalia in 1977. On May 8, 1980, the WHO declared
smallpox as officially eradicated from the earth.
Despite eradication of natural smallpox, many experts fear that this disease could
be reintroduced into the population intentionally by terrorists.
As immunity is thought to diminish after ten years and mass
vaccinations ceased in the mid 1970s, the current world
population would likely be susceptible to the virus. As the
virus is very stable in aerosol droplets and it is possible that
terrorists could employ some type of spraying device to attack
an airport or other crowded building. Due to our highly mobile
society and the high transmissibility of smallpox, one single
case of smallpox would constitute a worldwide emergency.
Smallpox image from www.mos.org/ cst/article/5109/
POSSIBLE BIOTERRORISM SCENARIO
Following intelligence from overseas sources, the FBI issues a terrorist alert to
the city of Northwest, with a population of 1.5 million. Local authorities are instructed to
tighten security at large gatherings such as sporting events and music concerts. Health
officials are directed to report any unusual medical cases that could represent
bioterrorism. Twenty days after the alert, a 27-year old Caucasian man appears at a
local hospital emergency room with 106-degree fever and pustules concentrated on his
face and extremities. At least one member of his family had a similar ailment.
Twelve days earlier, the man had attended
a football game with over 80,000 people present.
In consideration of the terrorist alert, the man is
immediately placed in isolation and tested for
smallpox. A preliminary antibody-based test,
however, is negative. One hospital physician, who
had been in Central Africa ten years ago,
diagnosed the patient’s symptoms to be very
similar to cases of monkeypox he had treated.
The rest of the attending physicians
concurred. In fact, the man’s brother-in-law
worked at the local zoo that had recently received
several animals from a rainforest in the
Democratic Republic of the Congo. To
conclusively determine whether the man had
smallpox, resulting from a bioattack, or
monkeypox, which is much less contagious, fluid is taken from a large pustule on the
patient’s arm. This fluid is then immediately sent to the Centers for Disease Control in
Atlanta for PCR testing.
Monkeypox image from http://www.uwlax.edu/clinmicro/clinicalmicro.htm
See further information about smallpox and possible threats at http://www.mos.org/cst/article/5109/
Also read the outbreak of monkeypox in the United States in 2003
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5225a4.htm
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Facts about Isolation and Vaccination Strategy: How Public Health
Officials will Respond to a Smallpox Outbreak
CDC has a detailed plan to protect Americans against the use of smallpox as a
biological weapon. This plan includes the creation and use of special teams of health
care and public health workers. If a smallpox case is found, these teams will take steps
immediately to control the spread of the disease. Smallpox was wiped out through
specific public health actions, including vaccination, and these actions will be used
again.
• If a smallpox outbreak happens, public health officials will use television, radio,
newspapers, the Internet and other channels to inform members of the public about
what to do to protect themselves and their families.
• Officials will tell people where to go for care if they think they have smallpox.
• Smallpox patients will be isolated (kept away from other people who could get sick
from them) and will receive the best medical care possible. Isolation prevents the virus
from spreading to others.
• Anyone who has had contact with a smallpox patient will be offered smallpox
vaccination as soon as possible. Then, the people who have had contact with those
individuals will also be vaccinated. Following vaccination, these people will need to
watch for any signs of smallpox. People who have been exposed to smallpox may be
asked to take their temperatures regularly and report the results to their health
department.
• The smallpox vaccine may also be offered to those who have not been exposed, but
would like to be vaccinated. At local clinics, the risks and benefits of the vaccine will be
explained and professionals will be available to answer questions.
• No one will be forced to be vaccinated, even if they have been exposed to smallpox.
• To prevent smallpox from spreading, anyone who has been in contact with a person
with smallpox but who decides not to get the vaccine may need to be isolated for at
least 18 days. During this time, they will be checked for symptoms of smallpox.
• People placed in isolation will not be able to go to work. Steps will be taken to care for
their everyday needs (e.g., food and other needs).
For more information about what CDC will do in the case of a real smallpox outbreak,
see www.bt.cdc.gov/agent/smallpox/basics/outbreak.asp.
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Lab 22 DNA-based Screening for Smallpox as a Bio-defense Strategy
Objectives:
After completing this lab a student should be able to:
1. Describe the disease smallpox the symptoms, diagnosis, treatment, prevention
and mortality rate.
2. Evaluate the difference between Smallpox and Monkeypox.
3. Describe the concern for differentiating these two organisms and the methods
used for confirming or ruling out
4. Perform a test using electrophoresis and DNA samples
5. Analyze the information from the test results to detect the presence of Smallpox
DNA.
6. Synthesize and communicate the ramifications of the information concerning the
potential effects of an outbreak and suggest a means to control both monkeypox
and smallpox.
Lab Materials
 Gel Electrophoresis
 Power Supply
 buffer
 Micropipets and tips
 Waterbath (65oC)
 DNA fragment A-F
 UV light
 Safety goggles
 Gloves
 Instastain
DNA Fragments
A Lane 1 DNA Standard
Marker
B Lane 2 Smallpox positive
control (simulated)
C Lane 3 Smallpox negative
control (simulated)
D Lane 4 Monkeypox control
DNA sample
E Lane 5 Patient 1
(simulated)
F Lane 6 Patient 2 (simulated)
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Lab Procedures
Loading the Gel
1. The gels and DNA samples have been prepared for you. Put on gloves, goggles,
and lab coats.
2. Use the sample gel and sample stain solution in the front of the class to practice
loading the submerged gel. The accuracy in loading is essential to good clear
results. Do not puncture or tear the gel in the process. Suck up a sample of the
stain. Use the sample pipettes and lower the tip into the well. Squirt the stain into
the well. A fresh gel will be used for the experiment.
3. Place the gel on its bed in the electrophoresis chamber. Be sure that the wells
are at the negative end of the electrophoresis apparatus because the DNA
fragments will migrate to the positive end.
4. Fill the electrophoresis apparatus chamber with the required volume of diluted
buffer. _____________________buffer mls
5. The gel is referred to as a submarine gel because it is completely submerged
under the buffer for the electrophoretic separation.
6. The DNA samples A-F need to be heated in a 65C water bath for 2 minutes to
help separate the fragments.
7. Carefully load the samples in the wells. Be sure and load sample A-E in
consecutive order in the wells (sample size 35-38l). If the samples are loaded in
the incorrect order the evaluations of the results may cause erroneous and
harmful diagnoses. Use a separate micropipette tip for each sample.
Electrophoresis
8. After all the samples are loaded, snap the cover in the electrode terminals,
making sure the negative and positive indicators on the cover and apparatus are
properly oriented.
9. Insert the black wire into the black input plug of the power source (negative
input). Insert the red wire into the red input of the power source (positive output).
10. Set the power source at the required voltage ______________Volts for the
required time ________minutes.
11. Check for bubbles at the electrodes to assure that the current is flowing.
12. Allow the tracking dye to migrate 3.5-4 cm from the wells. This should assure
separation of DNA bands.
13. Turn off the power, unplug the power source, disconnect the leads and remove
the cover. Carefully remove the gel for staining.
Staining the gel
14. Wearing gloves moisten the surface of the gel with a few drops of the buffer.
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Lab 22 Results DNA-based Screening for Smallpox as a Bio-defense Strategy
Name______________________________
1. Briefly describe smallpox the symptoms, diagnosis, treatment, prevention and
mortality rate.
2. Why does the CDC suspect that small pox is a potential bioterrorism agent?
3. What is the difference between Smallpox and Monkeypox? Explain the particular
concern in telling them apart.
4. What were the test results DNA electrophoresis? Draw the results on the gel
below and describe what the results mean for each lane.
1
2
Lanes
3
4
Lane 1 DNA A
5
6
Lane 2 DNA B
Lane 3 DNA C
Lane 4 DNA D
Lane 5 DNA E
Lane 6 DNA F
5. Did the physicians respond using the recommended CDC Strategy for suspected
smallpox. What else should be communicated concerning this outbreak?
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