University of Balamand

Faculty of Medicine and

Medical Sciences

CVIR401
Lecture#2
Diagnostic Tests in Virology

Instructor: Samer Bazzi, PhD

Diagnostic Methods To Study Viruses
a) Inoculation of a susceptible host (Lab animal) with infectious
material  observe signs of disease or determine the LD50 .

b) Inoculation of fertile hen’s eggs used for influenza virus.

c) In vitro cell culture systems to measure a cytopathic effect and to determine the
tissue culture infectious dose 50% (TCID50).

d) Viruses can be directly demonstrated by electron microscopy or by examining

histological appearances of tissues using light microscopy to demonstrate the
presence of inclusion bodies.

e) Serological tests check for the presence or level of specific antibodies in the blood

e) Non-cultivable viruses can be directly demonstrated through nucleic acid detection

by molecular techniques which can quantify copies of virus genome (Hepatitis C
virus) per ml of blood.
Virus Isolation and Propagation
Four types of cell cultures are widely used for virus isolation:
1- Primary cells – derived directly from animal tissues; monkey kidney cells can be
passaged 1 or 2 times.

2- Semi-continuous cells – capable of a limited No. (50 maximum) of passages before

undergoing senescence; Human embryonic kidney (HEK) cells and fibroblasts.

3- Continuous cell lines – immortalized cells can be passaged without limits. Examples
are HeLa-CD4, Vero cells (monkey kidney epithelial cells), and Madin-Darby canine
kidney (MDCK) cells (canine kidney epithelial cells).

4- Hematopoietic cells, peripheral blood mononuclear cells (PBMCs) or umbilical cord

mononuclear cells.

 Primary cells support the growth/isolation of the widest range of viruses. However,
they are take more growth time than cell lines and have limited growth potential.

 Continuous cells are the easiest to handle, but the range of viruses supported for
growth is often limited.
Problems with cell culture usage for virus isolation

- Sensitivity largely depends on the specimen condition.

- Susceptible to bacterial contamination or to toxic substances which may be present in

the specimen.

- Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrheal viruses,
Parvovirus.
Visualizing Viruses
1- Light Microscopy
Detecting cytopathic effects (CPE)
- Evidence of viral infections can be detected by the
characteristic sign of inclusion bodies in cells
(masses of virions or viral proteins at assembly
site)
- Inclusion bodies can be detected in cytologic
specimens or in histologically-stained tissues and
can be located in nucleus or/and in cytoplasm.
Nuclear Cytoplasmic Both
VZV Poxviruses Measles
HSV Parainfluenza CMV (Owl’s eye
inclusions)
Adenoviruses Rabies virus
(Negri bodies in
brain-infected
neurons)

Polyomaviruses
Parvovirus B19
Visualizing Viruses
1- Indirect visualization of viruses by light microscopy
Detecting cytopathic effects (CPEs)
Growing virus may produce CPE such as:
1- Ballooning of cells

CPE of HSV-1 on the McCoy

cell line (ballooning of cells)

2- Syncytium formation
Respiratory Syncytial Virus
(RSV) Measles Virus

Staining of cells with

eosinophilic dye
Visualizing Viruses
1- Indirect visualization of viruses by light microscopy
Detecting non-CPEs
A virus in culture may not produce CPE but could still be detected by:
1- Hemadsorption- cells acquire the ability to stick to mammalian RBCs.

2- Interference with CPE induced by another virus for example Rubella virus does
not produce CPE but can block CPE induced by enteroviruses.

Mumps virus-induced syncytia

formation and haemadsorption of
erythrocytes onto the surface of
the cell sheet.
Visualizing Viruses Lassa virus
virions
2- Direct visualization of viruses by electron
microscopy
- 106 virus particles per ml required for visualization; around
50,000-fold magnification is normally used. Viruses may be
detected in the following specimens:
Feces (Rotavirus, Adenovirus, Astrovirus, Calicivirus,
Norwalk like viruses)
Vesicle Fluid (Herpes simplex virus, Varicella zoster virus)
Skin scrapings (Papillomavirus, Molluscum contagiosum)

Classical Immune electron

microscopy (IEM): the sample is Viral particles
Expressing viral Agglutination
treated with specific anti-sera before
antigens
being put up for EM. Viral particles
present will be agglutinated and thus
congregate together by the antibody.

Solid phase immune electron Anti-sera

(Serum- containing
microscopy (SPIEM): the grid is
antibodies against
coated with specific anti-sera. Virus viral antigens)
particles present in the sample will be
adsorbed onto the grid by the antibody.
Counting Viruses
Infectivity assays (Plaque forming assays [PFA])
- CPEs induced by viruses can be used to quantitate infectious virus particles by the plaque-
forming assay.
- Serial dilutions of a virus have been plated on confluent monolayer culture of cells, and liquid
medium is replaced by overlaying semi-solid agarose (0.5%).
- Cells are stained (5% crystal violet) after a period of time in which a single virus infects a cell and
produces new virus particles. New virions then infect surrounding cells causing lysis.
- White areas (plaques) reflect cells which have been killed
- Each plaque is the result of the presence of one infectious virus particle. Results provide a
concentration of infectious virus particles termed plaque-forming units/ml.
Counting Viruses
Infectivity assays (Focus forming assay [FFA])
- FFA is an immunostaining technique and a variation of the viral plaque assay.
- Instead of detecting the plaque formation after virus-induced cell lysis these assays detect
infected host cells and infectious virus particle prior to plaque formation.
- Different cell monolayer samples are infected with a serial dilution of the virus of interest. After
incubating the samples overnight the host cells built clusters of infected cells which could be
visualized with specific horse radish peroxidase (HRP) coupled antibodies against a viral antigen

- The technique allows the identification of single foci and the subsequent calculation of the viral
titer which is reported as focus forming unit (FFU)/ml.

Focus-forming assay for hepatitis C virus (HCV). Infected cells were stained with an HCV-
specific primary antibody followed by a horse radish peroxidase (HRP)-conjugated
secondary antibody and finally a substrate is added for color development.
Quantifying Viruses
Hemagglutination Assay

- Hemagglutinating viruses (i.e. influenza viruses) bind to sialic acid residues on

RBCs. A single virion can bind to several different RBCs, and an RBC can be
bound by multiple virions to form a large network/mesh/lattice, of cells and virions
that is easily visualized.
 Direct detection of viral antigens
A wide range of techniques can be used

Precipitation RIA
Immunofluorescence Lateral flow
assay
Agglutination
ELISA
Detection of Viral Antigens
Enzyme-linked immunosorbent assay (ELISA)
- ELISA can be used for the detection of viral antigens as well as for the detection of virus-
specific antibodies.
- The specificity and sensitivity of these immunoassays are high.
Rapid Diagnostic Tests Based on Detecting Viral Antigens
Respiratory Specimen (nasopharyngeal aspirate or
wash, tracheal aspirate or bronchoalveolar lavage)
Viruses detected: Adenovirus, Measles, Influenza A, RSV,
Parainfluenza
GI specimen: stool Lateral
Viruses detected: Rotaviruses, Adenoviruses,
Astroviruses, Caliciviruses Flow
Skin specimen: vesicle fluid Assay
Viruses detected: HSV, VZV
Blood specimen: serum or plasma
Viruses detected: CMV, HBV, HIV

Advantages
● Results available in few minutes to
hours with some techniques.
Potential Problems
● Reduced sensitivity and specificity
(can be as low as 20%) on stool or
lavage samples
Viral Nucleic Acid Detection Assays
In situ hybridization (i.e. FISH)
- A technique where viral nucleic acids are localized in cells or tissue sections using
labeled DNA or RNA probes.

- Morphological information is retained; the cellular or sub-cellular distribution of viral

nucleic acids can therefore be determined
The images below illustrate the presence of different viruses in different type of
cells/tissues. Non-radioactive detection using a biotin-labelled probe, streptavidin-alkaline
phosphatase conjugate, and a chromogenic substrate as well as fluorescent probes
Viral Nucleic Acid Detection Assays
Polymerase chain reaction (PCR)

- PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and
thus, it is an extremely sensitive technique.
- It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the
target nucleic acid sequence of interest.
- These oligonucleotides act as primers for the thermostable Taq polymerase. Repeated cycles
(usually 25 to 40) of denaturation of the template DNA (at 95oC), annealing of primers to their
complementary sequences (55oC), and primer extension (72oC) result in the exponential
production of the specific target DNA fragment.
- Detection and identification of the PCR product is usually carried out by agarose gel
electrophoresis, hybridization with a specific oligonucleotide probe, restriction enzyme analysis,
or DNA sequencing.
Viral Nucleic Acid Detection Assays
PCR

Quantitative real-time PCR with

fluorescent-labeled probes for
Detection of PCR products (amplicons) parvovirus B19
by an agarose gel electrophoresis after
ethidium bromide staining
DNA
Ladder

Cq or Ct value
bp
Viral Nucleic Acid Detection Assays
PCR
Advantages of PCR
– Extremely high sensitivity, detect down to one viral genome per sample volume
– Easy to set up
– Fast turn-around time

Disadvantages of PCR
– Extremely liable to contamination
– High degree of operator skill required
Application of PCR in Virology Diagnostics Labs

- Monitoring of response to anti-viral therapy (HIV, Hep C).

- Diagnosis during the seronegative window (HIV, Rabies).

- Diagnosis of viral infection in the newborn (HIV DNA), in immunosuppressed patients

who fail to produce detectable virus-specific antibody, and in patients with passively
acquired antibodies that might confuse serological results

- Detection of herpesviruses in cerebrospinal fluid (HSV, VZV) Virus Genotyping of

HTLV, HCV, HIV etc.

- Detection of viruses which can not be isolated in tissue culture (some GI or

respiratory viruses), which lead to low level of viremia, or for which electron
microscopy and antigen detection techniques are not available.
Nucleic acid detection for viral diagnosis
Target Specimen Application
HIV-DNA Leukocytes (L) Perinatal infection
HIV-RNA Plasma (P) Viral load, response to therapy
HSV CSF/ocular FL Encephalitis/retinitis
VZV CSF/ocular FL Meningitis/retinitis
CMV Blood, L or P Infection after organ transplant
EBV CSF/L or P CNS infection/lymphoma
HHV6,7 Blood/ CSF Infection/encephalitis
HHV8 Blood/pleural FL KS/Body cavity lymphoma
Adenovirus Blood/Resp. Sec Disseminated/Respiratory infection
Parvovirus Serum/A-FL B19 chronic/congenital infecion

FL=fluid; Sec= secretions; Resp=respiratory; A=amniotic

Nucleic acid detection for viral diagnosis

Target Specimen Application

JC virus CSF Leukoencephalopathy
BK virus Urine/plasma Renal/HSC transplant
Papilloma Genital sec. Cervical carcinoma
Enterovirus Blood/CSF Systemic infect/meningitis
HCV Plasma Infection/RTT
HBV Serum Active infection/RTT
Rabies Saliva, CSF Diagnosis of infection
Resp. viruses Resp. samples Detect non-cultivable agent
GI viruses Feces Detect non-cultivable agent

HSC=hematopoietic stem cell; RTT=response to treatment

Serological Methods in Virology
- Viral serology activities involve detection and quantification of specific antibodies targeted
against a virus.
Serological Methods in Virology Presence of haemagglutination-
inhibiting Abs in serum
Presence of neutralizing Abs in serum

Different classes of Abs (IgG, IgM) can help

in differentiating between a current infection
and immunity (measured by ELISA)
Serological Methods in Virology
Western Blot
- Western blot allows the detection of different virus-specific antibodies
Usefulness of serological methods

- For viruses such as rubella and hepatitis A, the onset of clinical symptoms
coincide with the development of antibodies. The detection of IgM or rising titers
of IgG in the serum of the patient would indicate active disease.
- However, many viruses often produce clinical disease before the appearance
of antibodies such as respiratory and diarrheal viruses. So in this case, any
serological diagnosis would be retrospective and therefore will not be that
useful.
- There are also viruses which produce clinical disease months or years after
seroconversion e.g. HIV and Rabies. In the case of these viruses, the mere
presence of antibody is sufficient to make a definitive diagnosis (unless patient
is previously vaccinated).
Problems with serological methods
False negative serological tests
• Mild local infections such as HSV genitalis may not produce a detectable humoral
immune response.
• Immunocompromised patients often give a reduced or absent humoral immune
response.

False positive serological tests

• Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV or
Japanese B encephalitis and Dengue virus.
• Patients with infectious mononucleosis and those with connective tissue diseases
(SLE) may react non-specifically to viral antigens.
• Patients given blood or blood products may give a false positive result due to the
transfer of antibody.