Laboratory diagnosis:

The selection of the appropriate specimen for analysis is often complicated because
several viruses may cause the same clinical disease.
Specimen collection: is the most important step in laboratory diagnosis. The specimen
collection has some critical parameters:
 Time of collection
Specimens should be collected early in the acute phase of infection, before the virus
ceases to be shed. For example, respiratory viruses may be shed for only 3 to 7 days,
and shedding may lapse before the symptoms cease. HSV and varicella-zoster virus
(VZV) may not be recoverable from lesions more than 5 days after the onset of
symptoms. In addition, antibody produced in response to the infection may block the
detection of virus.
The shorter the interval between the collection of a specimen and its delivery to the
laboratory, the greater the potential for isolating a virus. The reasons are that many
viruses are labile, and the samples are susceptible to bacterial and fungal overgrowth.

 Site and amount

The site should be appropriate for the virus that has to be studied, blister for small pox,
CSF for polio virus. The amount should be sufficient enough to conduct tests.

 Type of collection
Should be suitable for the given test, e.g. A culture media needs a viable/living virus.
Volume of specimen
Storage temperature: 2-8 0 C
Significant losses in infectious titers occur when enveloped viruses (e.g.,
HSV, VZV, influenza virus) are kept at room temperature or frozen at
−20° C. This is not a risk for non-enveloped viruses (e.g., adenoviruses,
enteroviruses).
Note: Rapid tests are sensitive but not specific, so we need to do an additional test in order to confirm
the disease.

Direct Methods:
Electron Microscopy: it can be used to detect and identify some viruses if sufficient viral
particles are present. The addition of virus-specific antibody to a sample can cause viral
particles to clump, thereby facilitating the detection and simultaneous identification of
the virus (immunoelectron microscopy). Enteric viruses such as rota-viruses can be
detected in stool by these methods.
Not sensitive
 Time consuming
Expensive and laborious

Culture of the virus:

Virus in cell culture frequently produces a characteristic cytopathic effect that can
provide presumptive identification. CPE is the change in the appearance of the virus
infected cell and can be a change in shape, size or fusion to form a Syncytia [are
multinucleated giant cells formed by viral fusion of individual cells.] Paramyxoviruses,
HSV, VZV, and human immunodeficiency virus (HIV) promote syncytia formation.
Inclusion bodies are either histologic changes in the cells caused by viral components or
virus-induced changes in cell structures.

A type of cell in which virus produces a CPE are important clues in presumptive
identification.
Disadvantages: Sensitive but not specific.
Virus needs living cells.
High cost and time consuming.
Many clinically relevant viruses cannot grow in-vitro.

Viral Isolation and cell culture: A virus can be grown in tissue culture, embryonated eggs,
and experimental animals. Experimental animals are rarely used in clinical laboratories
for the purpose of isolating viruses.

Specific types of tissue culture cells are used to grow viruses.

Primary cell cultures are obtained by dissociating specific animal organs with
trypsin or collagenase. The cells obtained by this method are then grown as monolayers
(fibroblast or epithelial) or in suspension (lymphocyte) in artificial media supplemented
with bovine serum or another source of growth factors. Primary cells can be dissociated
with trypsin, diluted, and allowed to grow into new monolayers (passed) to become
secondary cell cultures.

Diploid cell lines are cultures of a single cell type that are capable of being passed
a large but finite number of times before, they senesce or undergo a significant change
in their characteristics.

Tumor cell lines and immortalized cell lines, usually initiated from human or
animal tumors or by treatment of primary cells with oncogenic viruses or chemicals,
consist of single cell types that can be passed continuously without senescing.
 Primary monkey kidney cells are excellent for the recovery of influenza viruses,
paramyxo-viruses, many entero-viruses and some adeno-viruses.

Human fetal diploid cells, which are generally fibroblastic cells, support the
growth of a broad spectrum of viruses such as HSV, VZV, CMV, adeno-viruses and
picorna-viruses.

HeLa cells, a continuous line of epithelial cells derived from a human cancer, are
excellent for the recovery of respiratory syncytial virus, adenoviruses, and HSV. Many
clinically significant viruses can be recovered in at least one of these cell cultures.

Disadvantages: Some viruses grow slowly or not at all or do not readily cause CPE in cell
lines typically used in clinical virology laboratories.

Detection of Viral Proteins: Enzymes and other proteins are produced during viral
replication and can be detected by biochemical, immunologic, and molecular biologic
means. Viral antigens on the cell surface or within the cell can be detected by
immunofluorescence and enzyme immunoassay (EIA).

DETECTION OF VIRAL GENETIC MATERIAL:

The genetic sequence of a virus is a major distinguishing characteristic of the family, type
and strain of virus. Different strains of HSV-1 and HSV-2 can be distinguished in this way
by restriction fragment length polymorphism.
DNA probes with sequences complementary to specific regions of a viral genome, can be
used like antibodies as sensitive and specific tools for detecting a virus. These probes can
detect the virus even in the absence of a viral replication. DNA probe analysis is
especially useful for detecting slowly replicating or non-productive viruses such as CMV
and Human Papilloma-Virus, or when the viral antigen cannot be detected using
immunologic tests. Specific viral genetic sequences in fixed, permeabilized tissue biopsy
specimens can be detected by in situ hybridization (fluorescence In-situ hybridization =
FISH).

Viral genomes can also be detected in clinical samples with the use of dot blot or
Southern blot analysis. For the latter method, the viral genome or electrophoretically
separated restriction endonuclease cleavage fragments of the genome are blotted onto
nitrocellulose filters and then detected on the filter by their hybridization to DNA probes.
Electrophoretically separated viral RNA (Northern blot– RNA: DNA probe hybridization)
blotted onto a nitrocellulose filter can be detected in a similar manner. The DNA probes
are detected with autoradiography or with fluorescent or EIA-like methods.

For many laboratories, the method of choice for detection, quantification, and
identification of viruses uses genome amplification techniques, including PCR for DNA
genomes and reverse transcriptase PCR (RT-PCR) for RNA genomes.

This technique is especially useful for detecting latent and integrated sequences of viruses,
such as retro-viruses, herpes-viruses, papilloma-viruses, and other papova-viruses.

For many laboratories, genome amplification techniques, including PCR for DNA
genomes and reverse transcriptase PCR (RT-PCR) for RNA genomes, are the method of
choice for detection and identification of viruses. Use of the appropriate primers for PCR
can promote a million fold amplification of a target sequenceRT- in a few hours. PCR
uses the retroviral reverse transcriptase to convert viral RNA to DNA and allow PCR
amplification of the viral nucleic acid sequences.

Advantages:
 Highly sensitive and specific
 Low cost
 Universally applicable
 Useful for HCV, HBV, Corona, Ebola virus & HIV diagnosis
Disadvantages:
 Can’t detect unknown viruses
 False positive in many viral infections

Analysis of the amplified viral nucleic acids is also being performed using matrix-assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Indirect Method:
Serological procedures: Serologic studies are used for the identification of viruses that
are difficult to isolate and grow in cell culture, as well as viruses that cause diseases of
long duration. Serology can be used to identify the virus and its strain or serotype,
whether it is an acute or chronic disease, and determine whether it is a primary infection
or a reinfection. The detection of virus-specific immunoglobulin M (IgM) antibody, which
is present during the first 2 or 3 weeks of a primary infection, generally indicates a recent
primary infection.

A rise in the titer of antibody can be used as diagnosis. A serum sample is collected as
soon as the virus etiology is suspected (acute phase) and a second sample is obtained 10-
14 days later(convalescent phase), if the antibody titer in the second sample is at least 4
times higher the patient is considered to be infected.

It includes: ELISA/Western Blot

Immunofluorescence essay
HA/HI etc.

Note: Monoclonal antibodies are used for diagnosis.

A. Enzyme-Linked Immunosorbent Assay

Enzyme immunoassay, which has many variations, depends on the conjugation of an
enzyme to an antibody. The enzyme is detected by assaying for enzyme activity with its
substrate. To measure antibody, known antigens are fixed on a solid phase (eg, plastic
microtiter plate), incubated with test antibody dilutions, washed, and reincubated with
an anti-immunoglobulin labeled with an enzyme (e.g., horseradish peroxidase). Enzyme
activity, measured by adding the specific substrate and estimating the color reaction, is
a direct function of the concentration of antibody bound. This serologic test is used to
detect antibodies to a number of infectious diseases, such as antibodies to HIV proteins
in blood samples or antibodies to the syphilis organism, Treponema pallidum.

The ELISA test is used to screen the blood supply to exclude individuals who are
seropositive for hepatitis B and C viruses and HIV.

B: Western Blotting:
Western blot analysis (also known as immunoblotting) is used to detect a specific
protein in a cell, tissue, organ, or body fluid. The technique depends on the reaction of
an antibody with a protein that is immobilized on a thin membrane. The ability of the
patient’s antibody to recognize specific viral proteins separated by electrophoresis,
transferred (blotted) onto a filter paper (e.g., nitrocellulose, nylon), and visualized with
an enzyme-conjugated antihuman antibody confirms the ELISA-indicated diagnosis of
HIV infection.
Advantages: Rapid and sensitive assay.
Safe and suitable for common screening (HIV)
Can give false positives.

Mechanism: Substrate of enzyme is added and amount of bound enzyme is determined.

Neutralization and HI tests assay antibody on the basis of its recognition of and binding
to virus. The antibody coating of the virus blocks its binding to indicator cells.
Antibody neutralization of virus inhibits infection and subsequent cytopathologic effects
in tissue culture cells. For HI, antibody in serum prevents a standardized amount of virus
from binding to and agglutinating erythrocytes.
The indirect fluorescent antibody test and solid-phase immunoassays, such as latex
agglutination and ELISA, are commonly used to detect and quantitate viral antigen and
antiviral antibody.

The Hemagglutination assay (typing and subtying of influenza virus) is used to detect and
quantify the concentration of virus required to cause blood agglutination. Viruses are
composed of a glycoprotein envelope which are able to bind to salic acid molecules
found on the receptor sites of red blood cells (RBC's) when cross links are formed
between the two, a lattice is produced which can be seen as agglutination.
HA:
 Hemagglutination protein of the influenza binds to saliac acid receptors on the
RBC.
 It only detects virus presence and antibody titer, not the strain
 In embryonated egg, it requires amplification of viral clinical specimen

HI:
Hemagglutination Inhibition Assay (HIA) is a procedure used to identify certain viruses
that can cause haemagglutination. This binding causes the formation of a lattice which
can be seen as agglutination.
 Measures the amount of patient’s antibody needs to inhibit agglutation of
RBC’s by virus.
 Antibody to heamagglutation can inhibit heamagglutation process, so more
virus is required to cause agglutation.
 Only viruses that agglutinate RBC can be detected.
 Both active and inactive viruses can be detected.

Advantages of Serological methods:

 Sensitive and specific
 Highly used
 Inexpensive except rapid tests
 Simple, easy to train

Disadvantages:
 Requires equipment and lab
 Indirect approach
 Dependent on host immunity