Laboratory diagnosis of viral infections is useful for the following purposes: To start antiviral drugs for those viral infections for which specific drugs are available such as herpes, CMV, HIV, influenza and respiratory syncytial virus (RSV) Screening of blood donors for HIV, hepatitis B and hepatitis C-helps in prevention of transfusion transmitted infections Surveillance purpose: To assess the disease burden in the community by estimating the prevalence and incidence of viral infections For outbreak or epidemic investigation, e.g. influenza epidemics, dengue outbreaks-to initiate appropriate control measures To start post-exposure prophylaxis of antiretroviral drugs to the health care workers following needle stick injury. To initiate certain measures: For example, If rubella is diagnosed in the first trimester of pregnancy, termination of pregnancy is recommended If newborn is diagnosed to have hepatitis B infection, then immunoglobulins (HBIG) should be started within 12 hours of birth.

1. LABORATORY DIAGNOSIS OF
VIRAL INFECTIONS
Insha jan

2. Laboratory diagnosis of viral infections
 Laboratory diagnosis of viral infections is useful for the
following purposes:
 To start antiviral drugs for those viral infections for which
specific drugs are available such as herpes, CMV, HIV, influenza
and respiratory syncytial virus (RSV)
 Screening of blood donors for HIV, hepatitis B and hepatitis C-
helps in prevention of transfusion transmitted infections
 Surveillance purpose: To assess the disease burden in the
community by estimating the prevalence and incidence of viral
infections
 For outbreak or epidemic investigation, e.g. influenza
epidemics, dengue outbreaks-to initiate appropriate control
measures

3. Laboratory diagnosis of viral infections
 To start post-exposure prophylaxis of antiretroviral drugs
to the health care workers following needle stick injury.
 To initiate certain measures: For example,
 If rubella is diagnosed in the first trimester of pregnancy,
termination of pregnancy is recommended
 If newborn is diagnosed to have hepatitis B infection, then
immunoglobulins (HBIG) should be started within 12 hours of
birth.

4. DIRECT DEMONSTRATION OF VIRUS
 Electron Microscopy
 Detection of viruses by electron microscopy (EM) is
increasingly used nowadays. Specimens are negatively
stained by potassium phosphotungstate and scanned
under EM.
 Shape: Viruses can be identified based on their distinct
appearances; for example:
 Rabies virus-bullet-shaped
 Rotavirus-wheel-shaped
 Coronavirus-petal-shaped peplomers
 Adenovirus-space vehicle-shaped
 Astrovirus-star-shaped peplomers.

5. DIRECT DEMONSTRATION OF VIRUS
 Direct detection from specimens: Direct detection of
viruses by EM is preferred as primary tool for diagnosis in
the following situations:
 For viruses that are difficult to cultivate; for e.g.
 Agents of viral gastroenteritis such as rotavirus, coronavirus,
adenovirus, calicivirus from diarrheal stool
 Hepatitis A and E viruses from feces
 Cytomegalovirus from urine (infants).
Virus detection from tissue culture: EM can also be used for
detection of viral growth in tissue cultures
Drawbacks: EM is highly expensive, has low sensitivity with a
detection threshold of 107 virions/ mL.The specificity is also low.

6. DIRECT DEMONSTRATION OF VIRUS
 lmmuno-electron Microscopy
 The sensitivity and specificity of EM can be improved by
adding specific antiviral antibody to the specimen to
aggregate the virus particles which can be centrifuged.
The sediment is negatively stained and viewed under EM.

7. DIRECT DEMONSTRATION OF VIRUS
 Fluorescent Microscopy
 Direct immunofluorescence (Direct-IF) technique is employed
to detect viral particles in the clinical samples.
 Procedure: Specimen is mounted on slide, stained with specific
antiviral antibody tagged with fluorescent dye and viewed
under fluorescent microscope
 Clinical applications:
 Diagnosis of rabies virus antigen in skin biopsies, corneal
smear of infected patients
 Syndromic approach: Rapid diagnosis of respiratory infections
caused by influenza virus, rhinoviruses, respiratory syncytial
virus, adenoviruses and herpesviruses can be carried out by
adding specific antibodies to each of these viruses
 Detection of adenovirus from conjunctiva! smears

8. DIRECT DEMONSTRATION OF VIRUS
 Light Microscopy
 Light microscopy is useful in the following situations.
 Inclusion bodies: Histopathological staining of tissue sections
may be useful for detection of inclusion bodies which helps in
the diagnosis of certain viral infections e.g. Negri bodies
detection in brain biopsies of patients or animals died of rabies
 Immunoperoxidase staining: Tissue sections or cells coated
with viral antigens are stained using antibodies tagged with
horse radish peroxidise following which hydrogen peroxide
and a coloring agent (benzidine derivative) are added. The
color complex formed can be viewed under light microscope.

9. DETECTION OF VIRAL ANTIGENS
 Various formats are available for detection of viral antigens in
serum and other samples such as enzyme-linked
immunosorbent assay (ELISA), immunochromatographic test
(ICT), flow through assays, etc. Some important antigen
detection tests include:
 HBsAg and HBeAg antigen detection for hepatitis B virus
infection from serum.
 NSl antigen detection for dengue virus infection from serum
 p24 antigen detection for HIV infected patients from serum
 Rotavirus antigen detection from diarrheic stool
 CMV specific pp65 antigen detection in peripheral blood
leukocyte.

10. DETECTION OF VIRAL ANTIBODIES
 Antibody detection from serum is one of the most
commonly used method in diagnostic virology.
 Appearance of IgM antibody or a four-fold rise of titer of
lgG antibody indicates recent infection; whereas presence
of IgG antibody ( without a recent rise) indicates chronic
or past infection.
 Various techniques available are described below:
 Conventional DiagnosticTechniques
 These are less commonly used nowaday. Examples
include:
 Heterophile agglutination test ( e.g. Paul-Bunnell test for
Epstein-Barr virus)

11. DETECTION OF VIRAL ANTIBODIES
 Conventional DiagnosticTechniques
 Hemagglutination inhibition (HAI) test for influenza virus
and arbovirus infection
 Neutralization test (for poliovirus and arbovirus
infections)
 Complement fixation test or CFT (for poliovirus,
arbovirus and rabies virus infections)

12. DETECTION OF VIRAL ANTIBODIES
 Newer Diagnostic Formats Newer techniques such as
ELISA, ICT, flow through assays are widely used for
antibody detection against most of the viral infections, for
example:
 Anti-HBc, Anti-HBs and Anti-HBe antibodies in serum for
hepatitis B infection
 Anti-Hepatitis C antibodies in serum
 Antibodies against HIV-1 and HIV-2 antigens from serum
 Anti-Dengue IgM/IgG antibodies from serum.

13. MOLECULAR METHODS
 Advent of molecular techniques has eased the diagnosis
of viral infections. They are more sensitive, specific and
yield quicker results than culture.
 Nucleic Acid Probe
 It is an enzyme or radio-labeled nucleic acid sequence
complementary to a part of nucleic acid sequence of the
target virus.
 When added to the clinical specimen, it hybridizes to the
corresponding part of viral nucleic acid

14. MOLECULAR METHODS
 Depending on the type of label attached to the probe, the
hybridized-labeled probe can be subsequently detected by
colorimetric methods (dot blot hybridization) or gamma
counting
 Both DNA and RNA probes are commercially available.
 Nucleic acid probes have a low sensitivity compared to
polymerase chain reaction (PCR) as it directly detects the
viral genes in the specimen, without amplification

15. MOLECULAR METHODS
 Polymerase Chain Reaction
 PCR has revolutionized the diagnostic virology.
 It involves three basic steps-(1) viral DNA extraction
from the specimen, (2) amplification of specific region of
viral DNA to 107 folds, (3) detection of amplified
products by gel electrophoresis.

16. MOLECULAR METHODS
 Reverse Transcriptase PCR (RT-PCR)
 RT-PCR is used for the detection of RNA viruses.
 After RNA extraction, the viral RNA is reverse
transcribed to DNA, which is then subjected to
amplification similar to that followed in PCR.
 Both PCR and RT-PCR cannot quantify the viral nucleic
acid load in the specimen

17. MOLECULAR METHODS
 Real Time PCR
 It has the advantage of quantifying viral nucleic acid in the
samples, hence used to monitor the treatment response,
e.g., monitoring the response to antiretroviral therapy.
 More so, it takes much less time than PCR as the
amplification is visualized on real time basis.

18. ISOLATION OF VIRUS
 Viruses cannot be grown on artificial culture media. They
are cultivated by animal inoculation, embryonated egg
inoculation or tissue cultures.
 Being labor intensive, technically demanding and time
consuming, virus isolation is not routinely used in
diagnostic virology
 The specimen should be collected properly and
immediately transported to the laboratory.
 Refrigeration is essential during transportation as most
viruses are heat labile.
 Type of specimen collected depends on the virus
suspected.

19. ISOLATION OF VIRUS
 Animal Inoculation
 Because of the ethical issues related to use of animals,
animal inoculation is largely restricted only for research
purpose.
 Research use: To study viral pathogenesis or viral
oncogenesis or for viral vaccine trials
 Diagnostic use: Primary isolation of certain viruses which
are difficult to cultivate otherwise; such as arboviruses
and coxsackieviruses

20. ISOLATION OF VIRUS
 Procedure: Infant (suckling) mice are used for the
isolation of viruses.
 Specimens are inoculated by intracerebral or
intraperitoneal routes.
 Mice are observed for signs of disease or death.
 Later on, they are sacrificed and the tissue sections are
subjected to histological examination
 Following intracerebral inoculation into suckling mice:
 Coxsackie-A virus produces flaccid paralysis
 Coxsackie-B virus produces spastic paralysis.

21. ISOLATION OF VIRUS
 Egg Inoculation
 Embryonated hen's eggs are used for cultivation of viruses.
 Eggs were first used for viral cultivation by Good pasture in
1931 and the method was further developed later by Burnet.
 Specimens can be inoculated by four different routes into
embryonated 7 to 12 days old hen's eggs and then incubated
for 2-9 days.

22. ISOLATION OF VIRUS
 Yolk Sac Inoculation
 It is preferred for arboviruses ( e.g.
Japanese B encephalitis virus, Saint
Louis encephalitis virus, and West
Nile virus) and some bacteria such
as Rickettsia, Chlamydia and
Haemophilus ducreyi. Growth of
the encephalitis viruses may result
in death of the embryo.
 Amniotic Sac
 It is mainly used for the primary
isolation of the influenza virus and
viral growth is measured by
detection of hemagglutinin antigens
in amniotic fluid.

23. ISOLATION OF VIRUS
 Allantoic Sac
 It is a larger cavity, hence is used for better yield of viral
vaccines. Example of egg derived vaccines are influenza vaccine,
yellow fever (17D) vaccine and Rabies (Flury strain) vaccine
Duck eggs are bigger than hen's eggs, therefore produce better
yield of rabies virus for preparation of inactivated non-neural
vaccine.

24. ISOLATION OF VIRUS
 Chorioallantoic Membrane
 It is preferred for poxviruses and other viruses such as HSV.
Viruses produce visible lesions Called as pocks on
chorioallantoic membrane (CAM).
 Pock counting: Each pock is derived from a single virion. So, the
number pocks would represent the number of viral particles
present in the inoculum Pocks produced by different viruses
have different morphology. For example,
 Vaccinia pocks are more hemorrhagic and necrotic than pocks of
variola virus
 Pocks ofHSV-2 are larger than HSV-1.
 Ceiling temperature: It is the maximum temperature above
which the pock formation is inhibited. Viruses vary in their
ceiling temperature, e.g. variola (37°C) and vaccinia ( 41 °C)

25. Tissue Culture
 Steinhardt was the first to use tissue culture in virology
(1913) who maintained the vaccinia virus in fragments of
rabbit cornea.
 Enders, Weller, and Robbins (1949) were able to culture
poliovirus in tissue cultures of non-neural origin and that
was the turning point following which tissue culture was
widely used in diagnostic virology.

26. Tissue Culture
 Tissue culture can be of three types:
 1. Organ culture: It was previously used for certain
fastidious viruses that have affinity to specific organs; for
example, tracheal ring culture for isolation of corona
virus.
 2. Explant culture: Fragments of minced tissue can be
grown as 'explants; e.g. adenoid explants used for
adenoviruses.This method is obsolete now.
 3. Cell line culture: This is the only isolation method
which is in use now. The preparation of cell lines and the
types of cell lines have been described below.

27. Tissue Culture
 Preparation of the Cell Lines
 Tissues are completely digested by treatment with
proteolytic enzymes (trypsin or collagenase), followed by
mechanical shaking so that the components are
completely dissociated into individual cells.
 Viral growth medium: The cells are then washed, counted,
and suspended in viral growth medium which contains
balanced salt solution added with essential amino acids
and vitamins, salts and glucose supplemented by 5-10% of
fetal calf serum and antibiotics.
 Medium is buffered with bicarbonate to maintain a pH of
7.2-7.4 and phenol red is added as pH indicator

28. Tissue Culture
 Tissue culture flasks: The viral growth medium containing
cells is dispensed in tissue culture flasks.
 Monolayer sheet formation: On incubation, the cells
adhere to the glass surfaces of the flask and then they
divide to form a confluent monolayer sheet of cells within
a week covering the floor of tissue culture flask

29. Tissue Culture
 Incubation: Tissue culture flasks are incubated horizontally
in presence of CO2 , either as a stationary culture or as a
roller drum culture.
 Rolling of the culture bottle in roller drums provides
better aeration which is useful for isolation of fastidious
viruses ( e.g. rotavirus).

30. Tissue Culture
 Types of Cell Lines
 The cell line cultures can be classified into three types
based on their origin, chromosomal characters, and
maximum number of cell divisions that they can undergo
 Primary cell lines: They are derived from normal cells
freshly taken from the organs and cultured.
 They are capable of very limited growth in culture,
maximum up to 5-10 divisions
 They maintain a diploid karyosome

31. Tissue Culture
 Useful for both primary isolation as well as growth of the
viruses for vaccine production
 Common examples include:
 Monkey kidney cell line- useful for isolation of myxoviruses,
enteroviruses and adenoviruses
 Human amnion cell line
 Chick embryo cell line.

32. Tissue Culture
 Secondary or diploid cell lines: They can divide maximum
up to 10-50 divisions before they undergo senescence
(death).
 They are also derived from the normal host cells and they
maintain the diploid karyosome.
 Common examples: Diploid cell lines are derived from
human fibroblasts and are useful for isolation of some
fastidious viruses as well as for viral vaccine preparation.

33. Tissue Culture
 Human fibroblast cell line: It is
excellent for the recovery of CMV.
 MRC-5 and WI-38 (human embryonic
lung cell strain):
 Used for preparation of various viral
vaccines, e.g. vaccines for rabies,
chickenpox, hepatitis-A and MMR
vaccines.
 They also support the growth of
spectrum of viruses ( e.g. HSV, VZV,
CMV, adenoviruses, and
picornaviruses ).

34. Tissue Culture
 Continuous cell lines
 They are derived from cancerous cell lines, hence are
immortal (capable of indefinite growth).
 They also possess altered haploid chromosome.
 They are easy to maintain in t he laboratories by serial
subculturing for indefinite divisions.
 This is the reason why continuous cell lines are the most
widely used cell lines.

35. Tissue Culture
 Common examples include :
 Hela cell line (Human carcinoma of
cervix cell line)
 HEp-2 cell line (Human epithelioma of
larynx cell line)- widely used for RSV,
adenoviruses and HSV
 KB cell line (Human carcinoma of
nasopharynx cell line)
 McCoy cell line (Human synovial
carcinoma cell line)- useful for isolation
of viruses, as well as Chlamydia
 Vero cell line (Vervet monkey kidney
cell line)-used for rabies vaccine
production
 BHK cell line (Baby hamster kidney cell
line).

36. Tissue Culture
 Detection ofViral Growth in Cell Cultures
 Following methods are used to detect the growth of the
virus in cell cultures.
 Cytopathic Effect (CPE): It is defined as the morphological
change produced by the virus in the cell line detected by
light microscope.
 Cytopathic viruses: Not all, but few viruses can produce
CPE and those are called as cytopathic viruses.
 The type of CPE is unique for each virus and that helps
for their presumptive identification.

37. Tissue Culture
 Viral Interference
 The growth of a non-CPE virus in cell culture can be
detected by the subsequent challenge of the cell line with
a known CPE virus. The growth of the first virus would
inhibit infection by the second virus by a mechanism
known as viral interference.
 For example, rubella is a non-CPE virus but prevents the -
replication of enteroviruses which are known to produce
CPE.

38. Tissue Culture
 Hemadsorption
 Hemagglutinating viruses (e.g. influenza virus) when
grown in cell lines, they produce hemagglutinin antigens
which are coated on the surface of the cell lines and can
be detected by adding guinea pig erythrocytes to the
cultures.
 The process of adsorption of erythrocytes to the surfaces
of infected cell lines is known as hemadsorption.

39. Tissue Culture
 Direct lmmunofluorescence Assay
 Virus infected cells are mounted on a slide and stained
with specific antibodies tagged with fluorescent dye and
viewed under fluorescent microscope for the presence of
viral antigens on the surface of infected cells.
 lmmunoperoxidase Staining
 Cells coated with viral antigens are stained by
immunoperoxidase tagged specific antibodies and viewed
under light microscope.

40. Tissue Culture
 Electron Microscopy
 The viruses can also be demonstrated in infected cell
lines by EM.
 Viral Genes Detection
 The presence of specific viral genes in culture fluid can be
detected by using PCR or nucleic acid probes.

41. VIRAL ASSAYS
 Viral assays are used for quantification of viral particles,
which can be grouped into physical and biological
methods.
 Physical Methods
 All these methods, estimate the total virus count ( or
viral antigen or gene count) and cannot distinguish
between infectious and non-infectious virus particles.
 Real time PCR: It can determine the number of viral
genome copies in a sample.

42. VIRAL ASSAYS
 Antigen detection assay such as radioimmunoassays (RIA)
and ELISA can be standardized to quantitate the amount
of virus in a sample.
 However, these tests may detect free viral proteins that
are not assembled into particles
 Hemagglutination assay: It is an easy and rapid method of
quantitating hemagglutinating viruses ( e.g. influenza virus).
 The viral hemagglutinin antigens can agglutinate RBCs by
binding to the specific receptors.

43. VIRAL ASSAYS
 Electron microscopy: Virus particles can be counted
directly by visualizing under the electron microscope by
comparing with a standard suspension of latex particles of
similar size.
 Biological Methods
 Biological methods detect the infectious virions only.
 Both qualitative (end point biological assays) or
quantitative (plaque assay and pock assay) methods are
available.

44. VIRAL ASSAYS
 End Point Biological Assays
 These assays depend on the measurement of animal
death/lesion, or CPE produced in tissue culture when
serial dilutions of the viral suspension are inoculated into
animals or cells.
 The titer is expressed as the 50 percent infectious dose
(ID50), which is the highest dilution of virus that
produces the effect in 50% of the cells or animals
inoculated.

45. VIRAL ASSAYS
 Plaque Assay
 It is the most widely used assay for quantifying infectious
viruses.
 Mono layer of cell line is inoculated with suitable dilutions of
the virus.
 After allowing time for adsorption of virus, the cell line is
covered with an agar layer so the viruses would spread only to
the immediate surrounding cells, but the spreading of the virus
throughout the culture will be prevented.
 Multiple cycles of replication and cell killing produce a small
area of infection called plaque
 Plaque counting: As single plaque arises from a single infectious
virus particle, hence the number of plaques counted would
represent the quantitative infectivity titer of the virus
suspension.

46. VIRAL ASSAYS
 Pock Assay
 Certain viruses such as variola, vaccinia and herpes form
pocks on chorioallantoic membrane (CAM) of
embryonated eggs.
 Number of pocks on CAM represents the approximate
number of infectious viral particles present in the dilution
inoculated.