WEEK 12|MYCOLOGY AND VIROLOGY
CHAPTER 29: Laboratory Diagnosis of Viral Infections
• Laboratories can provide different levels of
services, based on the mission, financial
resources, and need.
• Laboratory scientists in a clinical virology
laboratory must be familiar with both
molecular and non-molecular tests for
identifying viral infections
• Large equipment needed for a full-service
virology laboratory includes laminar flow
BSC (Level I or II), fluorescence microscope,
refrigerated centrifuge, roller drum for
holding cell culture tubes during incubation
(slow rotation continually bathes the cells in
the medium), inverted microscope used to
examine cell monolayers growing attached
to the inside surface beneath the liquid
medium.
• Inverted microscope used to examine cell
monolayers growing attached to the inside
surface beneath the liquid medium. Note
that the objective is under the glass test
tube, facilitating observation of the cell
monolayer
Specimen Selection
• A number of different clinical specimens
are suitable for the diagnosis of viral
diseases.
• The clinical symptoms of diseases often
point to the target organ(s) involved, which
can help determine the most appropriate
specimen(s) to collect.
• The patient’s symptoms and history,
including recent travel, the season of the
year, and a presumptive diagnosis, help
determine the appropriate procedures to
be used to identify a viral agent.
Lecturer: Sir Nathan John Jumalon
• For optimal recovery, specimens for viral
isolation should be collected from the
affected site.
o ***Read Table 29-2 on Chapter 29 of
Mahon.
o Appropriate Specimens for Maximum
Recovery
o Table 29-2 lists recommended
specimens to be collected for viral
diagnosis according to the body site
affected. Incorrect or poor specimen
collection can result in a false-negative
diagnostic result.
o For example, secretions from the
respiratory mucosa are most
appropriate for viral diagnosis of
respiratory infections.
o Aspirates, or surface swabs, are usually
appropriate for lesions. If the intestinal
mucosa is involved, a stool specimen is
most appropriate.
• For systemic, congenital, or generalized
disease: specimens from multiple sites,
including blood (buffy coat), CSF, and the
portals of entry (oral or respiratory tract) or
exit (urine or stool), are appropriate.
• Enteroviruses can cause respiratory
infections and may be recovered from the
stool after the respiratory shedding has
ceased.
o Enteroviruses generally infect by
the fecal–oral route and target
the gastrointestinal epithelium early
during their life cycle. They can also
infect by the respiratory tract as in the
case of enterovirus
D68 and rhinoviruses.
• In addition, enteroviruses are a major cause
of aseptic meningitis and can also be
isolated from urine specimens.
Specimen Collection
• Because viral shedding is usually greatest
during the early stages of infection, the best
specimens are those collected as early as
possible, which in many infections is even
before symptoms occur.
• Best specimens are collected as early as
possible
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WEEK 12|MYCOLOGY AND VIROLOGY Lecturer: Sir Nathan John Jumalon
CHAPTER 29: Laboratory Diagnosis of Viral Infections
• Specimens should be collected aseptically
• Aspirated secretions preferred
• Swabs are easier to use
o must be made of Dacron or rayon
o Rayon Tipped Swabs – Much like
cotton, rayon tipped swabs are
predictably soft and absorbent and an
economical choice for many
applications where cotton would not
be suitable. Rayon is a synthetic spun
fiber manufactured from wood pulp. It
is widely accepted for specimen
collection.
o Polyester Tipped Swabs – Polyester is a
synthetic spun fiber made from a
polymer. Originally introduced into the
realm of medical diagnostics by
DuPont under the brand name
Dacron®, it is now manufactured by
others and no longer carries a brand
name. Polyester fiber has been tested
and validated for use in specimen
collection in microbiology, rapid test
diagnostics and PCR analysis.
o Polyester tipped swabs boast excellent
collection and release properties and,
while somewhat more costly than
cotton or rayon, are not absorbent and
boast superior release properties.
Puritan purchases only the finest grade
spun polyester fibers which are then
produced to our specification of finish
and other characteristics to assure
reliable performance, every time.
• Do not use calcium alginate, charcoal, and Specimen Transport and Storage
swabs with wooden shafts
o Calcium alginate swabs inhibit the • Tissue samples must be kept moist
replication of some viruses and can o Viral transport medium
interfere with nucleic acid o Saline
amplification tests. o Trypticase soy broth
• standards for specimen collection. o Viral transport medium, saline, or
trypticase soy broth can be added to
sterile containers to keep tissues from
drying.
• Several viral transport systems are
commercially available
o Contains buffered isotonic solution with
protein such as albumin, gelatin, or
serum to protect less stable viruses.

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WEEK 12|MYCOLOGY AND VIROLOGY Lecturer: Sir Nathan John Jumalon
CHAPTER 29: Laboratory Diagnosis of Viral Infections
o Contains antibacterial and antifungal Direct Detection Methods Microscopy
agents – to inhibit contaminating
• In general, direct detection methods are
microorganisms.
not as sensitive as culture methods but can
• Samples that can be collected with viral
offer quick results to allow for rapid therapy.
transport media are respiratory, swab, and
Many of these tests can be performed in a
tissue samples.
few minutes.
• Samples that should be collected without
• Bright field light microscopy
viral transport media
o best for poxviruses; all other virus
o Blood
particles are too small to be seen.
o Bone marrow
• Electron microscopy
o CSF
o Greater magnification; can detect
o Amniotic fluid
virions
o Urine
o Expensive, labor-intensive, not very
o Pericardial and pleural fluids
sensitive
• The transport container should also be
o Rarely used in clinical laboratories
unbreakable and able to withstand
o Suited for large teaching or research
freezing and thawing.
institutions
• It is optimum to process viral specimens for
o Useful to detect non-culturable viruses
culture immediately. Process viral
such as the Norwalk viruses in stool
specimens for culture immediately (at best
filtrates.
within 12 to 24 hours of collection)
o If there is delay: store at 4°C, no more
than 5 days
o For longer delays: freeze at –70° C, 6 or Direct Detection Methods: Cytopathic Effect
more days (CPE)
• Many viruses are labile; Some viruses, such
• Distinct and characteristic visual changes in
as respiratory syncytial virus (RSV), become
infected cells
much more difficult to recover, even a few
• Detected in cell scrapings from infected
hours after collection.
sites via bright field microscopy
• Specimens should never be stored at –20° C
• Herpes simplex virus
because this temperature facilitates the
o Cytopathic effect: inclusion body
formation of ice crystals, which will disrupt
o Cowdry Type A bodies
the host cells and result in loss of viral
▪ Intranuclear eosinophilic droplet
viability.
like bodies

Methods in Diagnostic Virology

The clinical laboratory uses four major methods

to diagnose viral infections:

1. Direct detection of the virus in clinical

specimens
• Microscopy
• Enzyme immunoassays
2. Nucleic acid–based detection
3. Isolation of viruses in cell cultures
4. Serologic assays to detect antibodies to
virus

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WEEK 12|MYCOLOGY AND VIROLOGY Lecturer: Sir Nathan John Jumalon
CHAPTER 29: Laboratory Diagnosis of Viral Infections
• Human papillomavirus
o Cytopathic effect: inclusion body
o HPV-associated koilocytes
▪ squamous cells with an enlarged
nucleus surrounded by a
nonstaining halo

• Measles virus
o is a type of giant multinucleate cell
found in hyperplastic lymph nodes
early in the course of measles and also
in HIV-infected individuals
o Cytopathic effect: Multinucleated
Giant Cell (Syncytia)
o Warthin–Finkeldey cell

• Rabies virus
o Cytopathic effect: inclusion body
o Negri bodies
▪ Eosinophilic cytoplasmic inclusions
in neurons

• For instance, a Tzanck smear can detect

Cowdry type A bodies from herpes simplex
virus (HSV) and varicellazoster virus (VZV)
lesions, and Papanicolaou (Pap) smears
can reveal human papillomavirus (HPV)–
associated koilocytes, which are squamous
cells with an enlarged nucleus surrounded
by a nonstaining halo. Rabies is sometimes
diagnosed by detecting Negri bodies,
which are eosinophilic cytoplasmic
inclusions in neurons.
• Cytomegalovirus
o Cytopathic effect: inclusion body
o Owl-eye inclusions
o H&E stained lung section showing
typical owls-eye inclusions:
Direct Detection Methods: Detection of
Viral Antigens
• Direct fluorescent antibody (DFA) tests: cells
from a patient are fixed to a microscope
slide and fluorescence-labeled antibodies
are added. If viral antigens are present in
the sample, the labeled antibody will bind
and fluorescence will be seen
microscopically.
• DFA: adenovirus, influenza viruses A and B,
measles virus, parainfluenza viruses (PIVs) 1
through 4, and RSV from respiratory
specimens, HSV-1 and HSV-2 and VZV from
cutaneous lesion material, and
cytomegalovirus (CMV) from blood.

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WEEK 12|MYCOLOGY AND VIROLOGY
CHAPTER 29: Laboratory Diagnosis of Viral Infections
• Enzyme Immunoassays (EIA): many EIA tests
for viral detection are commercially
available with most using multiwell
microtiter plate assays.
• Detects:
o RSV and Influenza A (respiratory
specimens)
o hepatitis B virus (HBV) and HIV-1(serum
or plasma)
o Enteric Adenoviruses (stool)
o HSV (cutaneous lesions and
conjunctival swabs)
• EIA tests are often less sensitive than cell
cultures or IF tests, so negative results are
confirmed with cell culture or IF or nucleic
acid–based tests.
Nucleic Acid-Based Detection
Hybridization assay|PCR assays|Flow cytometry
Advantages
• Faster TAT
• More sensitive than cell culture and DFA
• Can be quantitative
• Detect non-culturable viruses
• Detect multiple viruses simultaneously
• Characterize virus genetically
Lecturer: Sir Nathan John Jumalon
Disadvantages
• Expensive
• Need for specialized training
• More complex facilities
• Lack of FDA-cleared assays
Viral Isolation
• In clinical virology, isolating viruses is still the
gold standard against which all other
methods are compared.
• Three methods for the isolation of viruses
o cell culture (most common)
o animal inoculation (extremely costly) –
used only as a special resource and in
reference or research laboratories. For
example, certain coxsackie A viruses
require suckling mice for isolation of the
virus.
o and embryonated eggs (rarely used) –
isolation of influenza viruses is
enhanced in embryonated eggs, but
this is generally accomplished more
easily in cell culture
Cell Culture
• Cell cultures can be divided into three
categories—primary, low passage (or
finite), and continuous.
• Each laboratory must decide which cell
lines to use based on the spectrum of viral
sensitivity, availability, and cost. Optimally,
several different cell lines will be used for a
single specimen to recover different viruses
that might be present,
Cell Culture: Primary
• Primary cell cultures are obtained from
tissue removed from an animal.
• The tissue is finely minced and then treated
with an enzyme such as trypsin to disperse
individual cells further. The cells are then
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WEEK 12|MYCOLOGY AND VIROLOGY
CHAPTER 29: Laboratory Diagnosis of Viral Infections
seeded onto a surface to form a
monolayer, such as in a flask or test tube
• Only minimal cell division occurs.
• Cell viability is maintained via splitting or
passaging – through periodically removing
cells from the surface, diluting them, and
placing them into a new container
• Primary cell lines can only be passaged a
few times before new cells must be
obtained.
• An example of a commonly used primary
cell culture is primary monkey kidney (PMK)
cells.
Low Passage (Finite or Diploid)
• can divide, but passage is limited to 50
generations
• they are diploid, meaning that they contain
two copies of each chromosome.
• Diploid is the normal genetic makeup for
eukaryotic cells
• As the number of passages increase, these
cells become more insensitive to viral
infection. Human neonatal lung is an
example of a standard finite cell culture
used in diagnostic virology.
• Must have at least 75% of cells with same
karyotype as the normal cells
• Examples: W1-38 and MRC-5
Lecturer: Sir Nathan John Jumalon
in diagnostic virology. Both HEp2 and
A549 were developed from cancer
tissue obtained from patients during
treatment.
Viral Isolation: Cell Culture Cytopathic Effect
(CPE)
• Inspection of cultures
• Indicated by areas of dead or dying cells
• Inverted light or PCM under LPO
• CPEs include:
o Rounding
o Fusion or syncytial formation
o Destruction and lysis
o Clumping
o Giant multinucleated cells
o Vacuolation
o Granulation
• Examples of viral cytopathic effect of
enterovirus infection in cell culture.
o LEFT: Monolayer of normal unstained
monkey kidney cells in culture showing
cell monolayer (120×).
o RIGHT: Unstained monkey kidney cell
culture illustrating advanced enteroviral
cytopathic effect with cell rounding,
cell detachment and necrosis (3+ to 4+
cytopathic effects) (120×). Almost 100%
of the cells are affected, and most of
the cell sheet has come loose from the
wall of the culture tube.

Continuous (Heteroploid or Immortal)

• Infinite passage LEFT

• < 75% normal cells and > 25% of cells
possess abnormal karyotype
• Malignant tissues
• Examples: HeLa, Hep-2, KB, A-549, Vero
o HEp2 (derived from a human laryngeal
epithelial carcinoma), A549 (derived
from a human lung carcinoma), and
Vero (derived from monkey kidney) are
examples of continuous cell lines used RIGHT

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WEEK 12|MYCOLOGY AND VIROLOGY
CHAPTER 29: Laboratory Diagnosis of Viral Infections
• Cytopathologic effect of herpes simplex
virus (HSV) infection.
o LEFT: Uninfected Vero cells, an African
green monkey kidney cell line.
o RIGHT: HSV-1–infected Vero cells
showing rounded cells, multinucleated
cells, and loss of the monolayer. Arrows
point to syncytia.
Lecturer: Sir Nathan John Jumalon
the H protein on their surface;
therefore, the RBCs will visibly
agglutinate.
• Principle: Viruses produce virus-specific
hemagglutinins (H) in the monolayer which
combine with RBCs of certain animals
• (+) Plaques of red blood cells
• Influenza A and B
o Exhibit both
• Parainfluenza and mumps
o Exhibit hemadsorption

LEFT

RIGHT

• Because influenza viruses typically do not VIRAL DETECTION: Hemadsorption

produce CPE, a hemagglutination or
• Hemadsorption of erythrocytes to cells
hemadsorption test is done to detect these
infected with influenza viruses, mumps virus,
viruses. Cells infected with influenza virus
parainfluenza viruses, or togaviruses. These
express a viral hemagglutinin (H) protein on
viruses express a hemagglutinin on their
their surface that binds red blood cells
surfaces, which binds to erythrocytes of
(RBCs).
selected animal species.
• Little or no detectable CPE
o Hemadsorption
▪ In the hemadsorption test, a
suspension of RBCs is added to the
infected cell monolayer. If
influenza virus is present, the RBCs
will adsorb or stick to the infected
cells.
o Hemagglutination
▪ In the hemagglutination assay,
supernatant from the infected
monolayer containing influenza
virus is mixed with a suspension of
RBCs. Influenza viruses also have

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WEEK 12|MYCOLOGY AND VIROLOGY
CHAPTER 29: Laboratory Diagnosis of Viral Infections
Viral Isolation: Shell Vial Culture
• Rapid modification of conventional culture
• The shell vial culture technique can more
rapidly identify viruses than the traditional
cell culture method. Cells are grown on a
round coverslip in a shell vial. A shell vial is a
small, round, flat-bottomed tube, generally
with a screw cap. The shell vial is inoculated
with the clinical sample and then
centrifuged to promote viral absorption.
• The shell vial is incubated for 24 to 48 hours,
after which the coverslip is removed, and
the IF technique performed. Based on the
type of clinical specimen and suspected
viruses, a variety of fluorescent-labeled
antibodies can be used. A modification of
this procedure is to use flat-bottomed
microtiter plates.
Lecturer: Sir Nathan John Jumalon
Serology
• Viral serology detects circulating antibodies
to viruses after exposure.
• First, it measures host response rather than
directly detecting the virus.
• Second, the antibody-producing
capabilities of human hosts vary widely
(immunocompromised individuals)
• Third, the antibody level does not
necessarily correlate with the acuteness or
activity level of the infection, because this is
also host-dependent.
• Tests include: ELISA, Western Blot,
Complement fixation, Indirect
Immunofluorescence
• Uses:
o Diagnosis of infections with
nonculturable agents, such as hepatitis
viruses
o Determination of immune status in
regard to rubella, measles, VZV, HAV,
and HBV
o Monitoring of patients who are
immunosuppressed or have had
transplants
o Epidemiologic or prevalence studies
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