VIRAL DETECTION

(Diagnostic Virology)

Dr. Alice Alma C. Bungay

Department of Medical Microbiology
College of Public Health
University of the Philippines Manila
Introduction

 Describes the laboratory methods

used to detect viral infections
 Methods may include:
 Culture
 Direct detection-Antigen
 Direct detection-Nucleic Acid
 Detection-Antibody
Guidelines for Specimen Collection, Handling
and Transport

 Appropriateness of specimen – purpose of test

• “right” or correct specimen – e.g.. swab vs.
aspirate
• time of collection
- during acute phase
- before antibiotics are administered
• Specimens/samples must be collected with sterile
collection methods
 Adequacy of specimen –volume/amount
 depends on the test to be performed
 Adherence to the Universal Precautions
Guidelines for Specimen Collection, Handling
and Transport

 Use specimen containers that are sterile, watertight

and can be securely closed to prevent leakage
 Label the specimen container as to:
 Name of the patient
 Date and time of collection
 Examination request
 Transport as soon as possible (ASAP)
 ensure survival and isolation of organisms
 prevent overgrowth of competing organisms
 Transport specimens in sturdy, sealable and break
resistant receptacles or containers
Guidelines for Specimen Collection, Handling
and Transport

 If specimen cannot be transported immediately,

store at –70 oC or liquid nitrogen temperature (-180
oC)

 DO NOT FREEZE SPECIMENS.

 Generally, all solid specimens except stool are
transported in viral transport medium
1. to prevent specimens from drying
2. helps maintain viability between collection and inoculation
3. retards microbial growth
 Specimens should be accompanied by a completely
filled out request form.
Diagnostic Methods in Virology
 Antigen detection
1. direct examination

2. indirect examination

 Antibody detection = Serology

 Direct Examination

 Antigen Detection
 Electron Microscopy
 Light Microscopy
 Viral Genome Detection
Direct Examination
1. Antigen Detection immunofluorescence, ELISA , Latex
agglutination etc.
2. Electron Microscopy morphology of virus particles
immune electron microscopy
3. Light Microscopy histological appearance
inclusion bodies
4. Viral Genome Detection hybridization with specific:
nucleic acid probes
polymerase chain reaction ( PCR)
ELISA for HIV
Electron Microscopy
 There are two fundamental
types of electron microscope,
the transmission type (TEM) &
scanning electron
microscopes (SEM).

TEM  Although beautiful images

with the appearance of three
dimensions are produced by
the SEM, for practical
investigations of virus
structure, the higher
magnifications achievable with
SEM the TEM have proved to be of
most value.
Electron Microscopy
Electron Microscopy
Examples of Electron micrographs
 the absolute number of virus particles present in any
preparation (total count)
 the appearance / structure of the particles
Gross Pathology
Histopathology
Viral Particles-Inclusion Bodies
Viral genome detection
Polymerase Chain Reaction
 Indirect Examination

Cellculture
Egg inoculation
Animals
Indirect Examination
1. Cell Culture cytopathic effect (CPE)
hemabsorption
immunofluorescence
2. Eggs pocks on CAM
haemagglutination
inclusion bodies
3. Animals disease or death
Virus Isolation
Plaque assay
 Renato Dulbecco in 1952 was the
first to accurately quantify animal
viruses using a plaque assay -
dilutions of the virus are used to
infect a cultured cell monolayer,
which is then covered with soft agar
to restrict diffusion of the virus,
resulting in localized cell killing & the
appearance of plaques after the
monolayer is stained.

 Counting the number of plaques

directly determines the number of
infectious virus particles applied to
the plate.
Cytopathic Effect (CPE)

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)
Cytopathic Effect (CPE)

Syncytium formation in cell

culture caused by RSV (top),
and measles virus (bottom).
(courtesy of Linda Stannard, University of Cape
Town, S.A.)
Shell Vial technique
Shell Vial technique
Hemadsorption

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.)
Rapid Diagnosis Based on the
Detection of Viral Antigens
Nasopharyngeal Aspirate RSV
Influenza A and B
Parainfluenza
Adenovirus
Feces Rotaviruses
Adenoviruses
Astrovirus
Skin HSV
VZV
Blood CMV (pp65 antigenaemia test)
CMV pp65 antigenaemia test
Immunofluorescense
Serology
 Serology
Detection of rising titres of antibody between acute and
convalescent stages of infection, or the detection of
IgM in primary infection.

Classical Techniques Newer Techniques

1. Complement fixation tests (CFT) 1. Radioimmunoassay (RIA)

2. Haemagglutination inhibition tests 2. Enzyme linked immunosorbent assay (EIA)
3. Immunofluorescence techniques (IF) 3. Particle agglutination
4. Neutralization tests 4. Western Blot (WB)
5. Counter-immunoelectrophoresis 5. RIBA, Line immunoassay
Serology
Criteria for diagnosing Primary Infection

 4 fold or more increase in titer of IgG or total antibody

between acute and convalescent sera
 Presence of IgM
 Seroconversion
 A single high titer of IgG (or total antibody) - very
unreliable

Criteria for diagnosing Reinfection

 fold or more increase in titer of IgG or total antibody
between acute and convalescent sera
 Absence or slight increase in IgM
Typical Serological Profile After Acute
Infection
Complement Fixation Test

Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement

fixation obtained with acute and convalescent phase serum specimens,
respectively. (2-fold serum dilutions were used) The observed 4-fold increase is
significant and indicates recent infection.
Western Blot

HIV-1 Western Blot

 Lane1: Positive Control
 Lane 2: Negative Control
 Sample A: Negative
 Sample B: Indeterminate
 Sample C: Positive