Laboratory Diagnosis of Smallpox Virus

Laboratory Diagnosis of
Smallpox Viral Infection

Presented by
Walid Fouad, Dr. Ahmad Moussa, Hamd Allah
Mohammad, Ahmad Ali El Tayeb, Akram
Fathy, Ramy Ali, Moemen Gamil, and
Mahrous Sayed
Microbiology Department, Medical Research
Institute Alexandria University
Variola Virus
1. One of the largest known viruses in size
(200 x 250 nm).
2. Belongs to family Poxviridae, and genus
Orthopoxvirus.
3. Enveloped and having a one piece of a double-
stranded DNA genome.
4. Causes the smallpox disease - a systemic viral
infection with fever and a characteristic skin rash that
resulted in high mortality rates in the developing
countries.
:Smallpox Disease
5. In 1980, the World Health Organization (WHO)

General Assembly ratified the declaration of success
made by the Global Commission for the Certification
of Smallpox Eradication.
6. Despite the eradication of naturally occurring

smallpox and the availability of a vaccine, the
potential "weaponization" of variola virus continues
to pose a military threat.
Diagnosing a smallpox viral infection
A. CLINICAL DIAGNOSIS
B. LABORATORY DIAGNOSIS:
1. Sample Collection
2. Light Microscopy and Histopathology
exam of stained smears
3. Electron Microscopy of pustular fluid
4. Tissue Culture
5. Serological/Immunological Tests
a. ELISA
b. Immunofluroscence Test
c. Virus Neutralization Test
d. Complement Fixation Test
e. Heamagglutination-inhibition Test
6. Molecular Diagnostic Techniques
- PCR
A. Clinical Diagnosis
Initial diagnosis of a smallpox
infection is most likely based on a
history and physical examination
findings.
Smallpox is an illness
characterized by:
a) An acute onset of fever

(> 38.3°C).
b) A rash that usually follows the fever, and is characterized by
firm, deep seated vesicles or pustules (blisters filled with fluid) in
the same stage of development.
:Other symptoms may include
 Severe headache
 Backache
 Fatigue
 Malaise
 Vomiting
 Diarrhea
 Excessive bleeding (in some cases)
** Before a diagnosis of smallpox is made, the doctor will consider other
illnesses that can mimic the signs and symptoms of smallpox.
Some of these illnesses include:
Chickenpox, Monkeypox, Herpes Zoster (Shingles), and Impetigo.
:Smallpox vs Chickenpox
A disease that can still be confused with smallpox is

varicella (chickenpox).
Chickenpox is caused by the varicella-zoster virus, a

DNA virus that belongs to the family of Herpesviridae.
Similar to smallpox, chickenpox is transmitted via

respiratory secretions or contact with skin lesions, and
usually presents with an abrupt onset of a pruritic rash,
low-grade fever, and malaise.
:Differential Diagnosis
Chickenpox and smallpox can
be distinguished by several
methods, including the clinical
course and the characteristic
appearance of the skin
lesions.
(1) Unlike smallpox, the

distribution of varicella lesions
is centripetal with a greater
concentration on the trunk.
Chickenpox does not usually
affect the palms and soles.
.(:Differential diagnosis (cont
(2) Additionally, chickenpox pustules

are of varying size due to variations
in the timing of pustule eruption (asyn-
chronous progression), where new
lesions appear in crops every few days
and are seen in different stages of
maturation.
Smallpox pustules, on the contrary,

are all very nearly the same size
since the viral effect progresses more
uniformly (synchronous progression).
Once a clinical case of a smallpox

infection is observed, it is important to
confirm it using laboratory tests.
:Differentiation between Smallpox and Chickenpox
B. Laboratoy Diagnosis
1. Collection of a specimen:
Either pustular fluid or scabs can serve
as specimens for laboratory evaluation.
For suspected cases of hemorrhagic
smallpox, the doctor may sample fluid
from a spinal tap (lumbar puncture).
To collect specimens, use forceps to

pick off a scab, the blunt edge of a
scalpel to open vesicles or pustules, and
a cotton swab to absorb the fluid.
Deposit specimens in a blood collection
test tube with a rubber stopper, seal the
stopper with adhesive tape, and enclose
the tube in a watertight container.
:Sample Collection
Specimens should be collected
only by a recently vaccinated
individual wearing latex gloves
and a surgical face mask.
Laboratory examination must

be performed in a Biosafety
level IV facility.
It should be also performed

only in designated laboratories
with the appropriate training
and equipment.
:Light Microscopy of stained smears. 2
Microscopically, poxviruses produce characteristic cytoplasmic

inclusions, known as Guarnieri bodies (also called B-type
inclusions) that can be seen by a light microscope.
Guarnieri bodies are characterized by being:

a. Eosinophilic intracytoplasmic viral inclusions.
b. Sites of viral replication in the host cell.
c. Variable in size, paranuclear (along side the nucleus).
c. Usually found upon microscopic inspection of epithelial cells
of patients suspected of having a poxvirus infection
(e.g. smallpox, monkeypox or vaccinia).
:Guarnieri bodies under light microscope
Histological sections or smears of
clinical specimens on glass slide are
stained with Hematoxylin and
Eosin (H&E), and Guarnieri bodies
usually appear as pink blobs in the
cytoplasm of affected epithelial cells.
Unlike A-type inclusions (which are
more strongly eosinophilic and only
found in infections with certain
poxviruses), Guarnieri bodies are
found in virtually all poxvirus
infections, but the absence of
Guarnieri bodies cannot be used to
rule out smallpox.Examining affected
cells for Guarnieri bodies can not
help in differentiating a smallpox
viral infection from other poxvirus
infections.
:Electron Microscopy of pustular fluid. 3
The diagnosis of an orthopoxvirus infection
can also be made rapidly by electron
microscopic examination of pustular
fluid or scabs. EM can distinguish orthopox
viruses from other viral agents.
However, all orthopoxviruses exhibit

identical, smooth, rounded "brick-
shaped" virions by electron microscopy.
Thus, electron microscopy of vesicular

scraping will not help to differentiate
between variola, vaccinia, cowpox or
monkeypox viruses.
The definitive diagnosis and identification

of species of orthopoxvirus rests on viral
culture and characterization of the virus
by various biologic assays, including
PCR and restriction fragment-length
polymorphisms
Tissue culture technique . 4
:and examining pock morphology on CAMs
Using tissue culture techniques, a
smallpox infection can be definitely
diagnosed by:
1. Growing the virus on Chorio-

Allantoic Membrane - CAM (part of a
chicken embryo), and
2. Examining the resulting pock

lesions under defined temperature
conditions.
This method is very helpful in the

differential diagnosis between a
smallpox (variola) virus, and other
members of the genus Orthopoxvirus
(such as: monkeypox, vaccina, and
cowpox viruses).
:Viral Pock Morphology
When a chorioallantoic membrane of 12-day-old developing
chicken embryos are inoculated with orthopoxviruses and
incubated at 35 C for 72 hours, morphologically
characteristic pocks typical of each virus will be
observed as shown in the following figures:
a) Variola (smallpox) virus produces small, grey-

white pocks.
b) Vaccinia virus produces larger, opaque, white

pocks with pale hemorrhagic ulcer.
c) Monkeypox virus produces small hemorrhagic

ulcer.
d) Cowpox virus produces hemorrhagic pocks.

:Serological/Immunological Tests. 5
I. Enzyme linked immunosorbent assay (ELISA):
a) ELISA can be used to detect variola virus-specific immunoglobulins.
b) It may also used to detect a specific viral antigen by using the “double antibody
Sandwich” technique.
II. Immunofluroscence Test:

a) Relatively simple and rapid.
b) Used for detecting viral (antigen) particles in suspected samples.
c) In the indirect immunofluroscence technique, an immune rabbit serum is used to treat the
sample smears. This is followed by the addition of an anti-rabbit serum "coupled" with a
fluorescent dye. A fluoresencent sample means a positive result.
c) The indirect technique gives more reliable results than the direct one.
:ELISA procedures
.(:Serological Tests (cont
III. Viral Neutralization Test (VNT):
a) Definition - Neutralization of a virus is defined as the “loss of infectivity through reaction of the virus
with its specific antibody”.
b) Purpose - Neutralization test is used to determine the antiviral activity of the serum of a suspected
case. (I.e.In this test, the serum of the patient is tested for its power to "neutralize" variola virus in tissue culture
or on CAM).
C) Test Steps - 1) Mixing the virus with the serum to be tested (which is supposed to contain virus-
neutralizing antibodies) under appropriate condition, and then 2) Inoculating that mixture into cell culture,
eggs or animals. Neutralization test, therefore, requires that an active suspension of that virus should be at
hand.
:Disadvantages of Neutralization Test**
a) The test is rather more time-consuming than complement-fixation or heamagglutination inhibition.
b) The results are not always easy to interpret if the patient has been previously vaccinated against
smallpox. Neutralizing antibodies may persist in serum for years after smallpox vaccination, and usually
fade in about 10 years.
IV. Complement Fixation Test (CFT):

The complement fixation test is an immunological medical test that can be used to detect the presence
of either specific antibody or specific antigen in a patient's serum.
:Methodology of the Complement Fixation Test
V. Heamagglutination Inhibition Test (HIT):

A wide variety of different viruses possess the ability to agglutinate
erythrocytes. This is the outcome of the haemagglutinin part of the viral protein
binding to receptors on the membrane of red blood cells. The linking together of the
red blood cells by the viral particles results in clumping. This clumping is known as
haemagglutination, and is visible macroscopically and is the basis of
haemagglutination tests to detect the presence of viral particles.
Antibodies against the viral protein responsible for haemagglutination can prevent
haemagglutination; this is the basis behind the haemagglutination-inhibition test
(HAI).
 The HAI test is simple to

perform.
 Serial dilutions of patient's sera

are allowed to react with a fixed
dose of viral haemagglutinin,
followed by the addition of
agglutinable erythrocytes.
 In the presence of antibody, the

ability of the virus to agglutinate
the erythrocytes is inhibited.
:Molecular Diagnostic Techniques. 6
Polymerase Chain Reaction (PCR):

a) PCR is a relatively simple, rapid, sensitive, and accurate method for detection
and differential diagnosis of several different orthopoxvirus infections.
b) PCR helps in diagnosing a smallpox infection through the identification of

species-specific DNA sequences.
d) Various polymerase chain reaction techniques:
- Essential conserved genes (E9L, A25R) - difficult to discriminate among

species of orthopoxviruses.
- Nonessential, variable genes (HA, HTI, crmB) - species specific.

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Laboratory Diagnosis of Smallpox Virus

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Laboratory Diagnosis of

Smallpox Viral Infection

5. In 1980, the World Health Organization (WHO)

6. Despite the eradication of naturally occurring

a) An acute onset of fever

A disease that can still be confused with smallpox is

Chickenpox is caused by the varicella-zoster virus, a

Similar to smallpox, chickenpox is transmitted via

(1) Unlike smallpox, the

(2) Additionally, chickenpox pustules

Smallpox pustules, on the contrary,

Once a clinical case of a smallpox

To collect specimens, use forceps to

Laboratory examination must

It should be also performed

Microscopically, poxviruses produce characteristic cytoplasmic

Guarnieri bodies are characterized by being:

However, all orthopoxviruses exhibit

Thus, electron microscopy of vesicular

The definitive diagnosis and identification

1. Growing the virus on Chorio-

2. Examining the resulting pock

This method is very helpful in the

a) Variola (smallpox) virus produces small, grey-

b) Vaccinia virus produces larger, opaque, white

c) Monkeypox virus produces small hemorrhagic

d) Cowpox virus produces hemorrhagic pocks.

II. Immunofluroscence Test:

III. Viral Neutralization Test (VNT):

a) The test is rather more time-consuming than complement-fixation or heamagglutination inhibition.

IV. Complement Fixation Test (CFT):

V. Heamagglutination Inhibition Test (HIT):

 The HAI test is simple to

 Serial dilutions of patient's sera

 In the presence of antibody, the

Polymerase Chain Reaction (PCR):

b) PCR helps in diagnosing a smallpox infection through the identification of

d) Various polymerase chain reaction techniques:

- Essential conserved genes (E9L, A25R) - difficult to discriminate among

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